Polyalkylene oxide-modified single chain polypeptides

ABSTRACT

The present invention relates to the chemical modification of single chain polypeptides by means of covalent attachment of strands of poly(ethylene glycol) PEG and similar poly(alkylene oxides) to single chain polypeptide binding molecules that have the three dimensional folding and, thus, the binding ability and specificity, of the variable region of an antibody. Such preparations of modified single chain polypeptide binding molecules have reduced immugenicity and antigenicity as well as having a longer halflife in the bloodstream as compared to the parent polypeptide. These beneficial properties of the modified single chain polypeptide binding molecules make them very useful in a variety of therapeutic applications. The invention also relates to multivalent antigen-binding molecules capable of PEGylation. Compositions of, genetic constructions for, methods of use, and methods for producing PEGylated antigen-binding proteins are disclosed.

[0001] This application claims the benefit of the filing date of each ofthe following applications: No. 60/044,449 filed Apr. 30,1997;60/050,472 filed Jun. 23, 1997; No. 60/063,074 filed Oct. 27, 1997; andNo. 60/067,341 filed Dec. 2, 1997, each of which is incorporated hereinby reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the chemical modification ofsingle chain polypeptides by means of covalent attachment of strands ofpoly(ethylene glycol) PEG and similar poly(alkylene oxides) to singlechain polypeptide binding molecules that have the three dimensionalfolding and, thus, the binding ability and specificity, of the variableregion of an antibody. Such preparations of modified single chainpolypeptide binding molecules have reduced immugenicity and antigenicityas well as having a longer halflife in the bloodstream as compared tothe parent polypeptide. These beneficial properties of the modifiedsingle chain polypeptide binding molecules make them very useful in avariety of therapeutic applications. The invention also relates tomultivalent antigen-binding molecules capable of PEGylation.Compositions of, genetic constructions for, methods of use, and methodsfor producing PEGylated antigen-binding proteins are disclosed.

[0004] 2. Description of Related Art

[0005] Antibodies are proteins generated by the immune system to providea specific molecule capable of complexing with an invading molecule,termed an antigen. Natural antibodies have two identical antigen-bindingsites, both of which are specific to a particular antigen. The antibodymolecule “recognizes” the antigen by complexing its antigen-bindingsites with areas of the antigen termed epitopes. The epitopes fit intothe conformational architecture of the antigen-binding sites of theantibody, enabling the antibody to bind to the antigen.

[0006] The antibody molecule is composed of two identical heavy and twoidentical light polypeptide chains, held together by interchaindisulfide bonds. The remainder of this discussion on antibodies willrefer only to one pair of light/heavy chains, as each light/heavy pairis identical. Each individual light and heavy chain folds into regionsof approximately 110 amino acids, assuming a conserved three-dimensionalconformation. The light chain comprises one variable region (V_(L)) andone constant region (C_(L)), while the heavy chain comprises onevariable region (V_(H)) and three constant regions (C_(H) ¹, C_(H) ² andC_(H) ³). Pairs of regions associate to form discrete structures. Inparticular, the light and heavy chain variable regions associate to forman “Fv” area which contains the antigen-binding site. The constantregions are not necessary for antigen binding and in some cases can beseparated from the antibody molecule by proteolysis, yieldingbiologically active (i.e., binding) variable regions composed of half ofa light chain and one quarter of a heavy chain.

[0007] Further, all antibodies of a certain class and their Fabfragments (i.e., fragments composed of V_(L), C_(L), V_(H), and C_(H) ¹)whose structures have been determined by x-ray crystallography showsimilar variable region structures despite large differences in thesequence of hypervariable segments even when from different animalspecies. The immunoglobulin variable region seems to be tolerant towardsmutations in the antigen-binding loops. Therefore, other than in thehypervariable regions, most of the so-called “variable” regions ofantibodies, which are defined by both heavy and light chains, are, infact, quite constant in their three dimensional arrangement. See forexample, Huber, R., Science 233:702-703 (1986)).

[0008] Recent advances in immunobiology, recombinant DNA technology, andcomputer science have allowed the creation of single polypeptide chainmolecules that bind antigen. These single-chain antigen-bindingmolecules (“SCA”) or single-chain variable fragments of antibodies(“sFv”) incorporate a linker polypeptide to bridge the individualvariable regions, V_(L) and V_(H), into a single polypeptide chain. Adescription of the theory and production of single-chain antigen-bindingproteins is found in Ladner et al., U.S. Pat. Nos. 4,946,778, 5,260,203,5,455,030 and 5,518,889. The single-chain antigen-binding proteinsproduced under the process recited in the above U.S. patents havebinding specificity and affinity substantially similar to that of thecorresponding Fab fragment. A computer-assisted method for linker designis described more particularly in Ladner et al., U.S. Pat. Nos.4,704,692 and 4,881,175, and WO 94/12520.

[0009] The in vivo properties of sFv (SCA) polypeptides are differentfrom MAbs and antibody fragments. Due to their small size, sFv (SCA)polypeptides clear more rapidly from the blood and penetrate morerapidly into tissues (Milenic, D. E. et al., Cancer Research51:6363-6371(1991); Colcher et al., J. Natl. Cancer Inst. 82:1191(1990); Yokota et al., Cancer Research 52:3402 (1992)). Due to lack ofconstant regions, sFv (SCA) polypeptides are not retained in tissuessuch as the liver and kidneys. Due to the rapid clearance and lack ofconstant regions, sFv (SCA) polypeptides will have low immunogenicity.Thus, sFv (S CA) polypeptides have applications in cancer diagnosis andtherapy, where rapid tissue penetration and clearance, and ease ofmicrobial production are advantageous.

[0010] A multivalent antigen-binding protein has more than oneantigen-binding site. A multivalent antigen-binding protein comprisestwo or more single-chain protein molecules. Enhanced binding activity,di- and multi-specific binding, and other novel uses of multivalentantigen-binding proteins have been demonstrated. See, Whitlow, M., etal., Protein Engng. 7:1017-1026 (1994); Hoogenboom, H. R., NatureBiotech. 15:125-126 (1997); and WO 93/11161.

[0011] Ladner et al. also discloses the use of the single chain antigenbinding molecules in diagnostics, therapeutics, in vivo and in vitroimaging, purifications, and biosensors. The use of the single chainantigen binding molecules in immobilized form, or in detectably labeledforms is also disclosed, as well as conjugates of the single chainantigen binding molecules with therapeutic agents, such as drugs orspecific toxins, for delivery to a specific site in an animal, such as ahuman patient.

[0012] Whitlow et al. (Methods: A Companion to Methods in Enzymology2(2):97-105 (June, 1991)) provide a good review of the art of singlechain antigen binding molecules and describe a process for making them.

[0013] In U.S. Pat. No. 5,091,513, Huston et al. discloses a family ofsynthetic proteins having affinity for preselected antigens. Thecontents of U.S. Pat. No. 5,091,513 are incorporated by referenceherein. The proteins are characterized by one or more sequences of aminoacids constituting a region that behaves as a biosynthetic antibodybinding site (BABS). The sites comprise (1) noncovalently associated ordisulfide bonded synthetic V_(H) and V_(L) regions, (2) V_(H)-V_(L) orV_(L)-V_(H) single chains wherein the V_(H) and V_(L) are attached to apolypeptide linker, or (3) individual V_(H) or V_(L) domains. Thebinding domains comprises complementarity determining regions (CDRs)linked to framework regions (FRs), which may be derived from separateimmunoglobulins.

[0014] U.S. Pat. No. 5,091,513 also discloses that three subregions (theCDRs) of the variable domain of each of the heavy and light chainsofnative immunoglobulin molecules collectively are responsible forantigen recognition and binding. These CDRs consist of one of thehypervariable regions or loops and of selected amino acids or amino acidsequences disposed in the framework regions that flank that particularhypervariable region. It is said that framework regions from diversespecies are effective in maintaining CDRs from diverse other species inproper conformation so as to achieve true immunochemical bindingproperties in a biosynthetic protein.

[0015] U.S. Pat. No. 5,091,513 includes a description of a chimericpolypeptide that is a single chain composite polypeptide comprising acomplete antibody binding site. This single chain composite polypeptideis described as having a structure patterned after tandem V_(H) andV_(L) domains, with a carboxyl terminal of one attached through an aminoacid sequence to the amino terminal of the other. It thus comprises anamino acid sequence that is homologous to a portion of the variableregion of an immunoglobulin heavy chain (V_(H)) peptide bonded to asecond amino acid sequence that was homologous to a portion of thevariable region of an immunoglobulin light chain (V_(L)).

[0016] The covalent attachment of strands of a polyalkylene glycol to apolypeptide molecule is disclosed in U.S. Pat. No. 4,179,337 to Davis etal., as well as in Abuchowski and Davis “Enzymes as Drugs,” Holcenbergand Roberts, Eds., pp. 367-383, John Wiley and Sons, New York (1981).These references disclosed that proteins and enzymes modified withpolyethylene glycols have reduced immunogenicity and antigenicity andhave longer lifetimes in the bloodstream, compared to the parentcompounds. The resultant beneficial properties of the chemicallymodified conjugates are very useful in a variety of therapeuticapplications.

[0017] Although amino acid sequences such as the single chainpolypeptides described above, and fusion proteins thereof, have not beenassociated with significant antigenicity in mammals, it has beendesirable to prolong the circulating life and even further reduce thepossibility of an antigenic response. The relatively small size of thepolypeptides and their delicate structure/activity relationship,however, have made polyethylene glycol modification difficult andunpredictable. Most importantly, it was unknown how to modulate retainedactivity of the polypeptides after conjugation with polymers, such asPEG.

[0018] To effect covalent attachment of polyethylene glycol (PEG) orpolyalkalene oxides to a protein, the hydroxyl end groups of the polymermust first be converted into reactive functional groups. This process isfrequently referred to as “activation” and the product is called“activated PEG” or activated polyalkylene oxide. Methoxy poly(ethyleneglycol) (mPEG), capped on one end with a functional group, reactivetowards amines on a protein molecule, is used in most cases.

[0019] The activated polymers are reacted with a therapeutic agenthaving nucleophilic functional groups that serve as attachment sites.One nucleophilic functional group commonly used as an attachment site isthe E-amino groups of lysines. Free carboxylic acid groups, suitablyactivated carbonyl groups, oxidized carbohydrate moieties and mercaptogroups have also been used as attachment sites.

[0020] The hydroxyl group of PEG has been activated with cyanuricchloride and the resulting compound is then coupled with proteins(Abuchowski et al., J. Biol. Chem. 252:3578 (1977); Abuchowski & Davis,supra (1981)). However, there are disadvantages in using this method,such as the toxicity of cyanuric chloride and its non-specificreactivity for proteins having functional groups other than amines, suchas free essential cysteine or tyrosine residues.

[0021] In order to overcome these and other disadvantages, alternativeactivated PEGs, such as succinimidyl succinate derivatives of PEG(“SS-PEG”), have been introduced (Abuchowski et al., Cancer Biochem.Biophys. 7:175-186 (1984)). SS-PEG reacts quickly with proteins (30minutes) under mild conditions yielding active yet extensively modifiedconjugates.

[0022] Zalipsky, in U.S. Pat. No. 5,122,614, discloses poly(ethyleneglycol)-N-succinimide carbonate and its preparation. This form of thepolymer is said to react readily with the amino groups of proteins, aswell as low molecular weight peptides and other materials that containfree amino groups.

[0023] Other linkages between the amino groups of the protein, and thePEG are also known in the art, such as urethane linkages (Veronese etal., Appl. Biochem. Biotechnol. 11:141-152 (1985)), carbarnate linkages(Beauchamp et al., Analyt. Biochem. 131:25-33 (1983)), and others.

[0024] Suzuki et al. (Biochimica et Biophysica Acta, 788: 248-255(1984)) covalently couples immunoglobulin G (IgG) to poly(ethyleneglycol) that has previously been activated by cyanuric chloride. Thecoupled IgG was studied for physicochemical and biological propertiessuch as molecular structure, size-exclusion chromatographic behavior,surface activity, interfacial aggregability, heat aggregability inducingnonspecific complement activation, and antigen-binding activity. Thepoly(ethylene glycol) coupling to IgG increased the apparent Stokes'radius and the surface activity of IgG and stabilized IgG on heatingand/or on exposure to interfaces, while no structural denaturation ofIgG was observed. The suppressed nonspecific aggregability wasinterpreted mainly by difficulty in association between the modified IgGmolecules. These results indicated the use of the poly(ethyleneglycol)-coupled IgG as an intravenous preparation and also as anadditive stabilizing intact IgG for intravenous use.

[0025] Sharp et al. (Analytical Biochemistry 154: 110-117 (1986))investigated the possibility of producing biospecific affinity ligandsfor separating cells in two polymer aqueous phase systems on the basisof cell surface antigens. Rabbit anti-human erythrocyte IgG was reactedwith cyanuric chloride-activated monomethyl poly(ethylene glycol)fractions (molecular weights approximately 200, 1900, and 5000) atvarious molar ratios of PEG to protein lysine groups. The partitioncoefficient of the protein in a Dextran/PEG two phase system increasedwith increasing degree of modification and increasing PEG molecularweight. There was a concomitant loss in ability to agglutinate humanerythrocytes.

[0026] Tullis, in U.S. Pat. No. 4,904,582, describes oligonucleotideconjugates wherein the oligonucleotides are joined through a linking armto a hydrophobic moiety, which could be a polyalkyleneoxy group. Theresulting conjugates are said to be more efficient in membranetransport, so as to be capable of crossing the membrane and effectivelymodulating a transcriptional system. In this way, the compositions canbe used in vitro and in vivo, for studying cellular processes,protecting mammalian hosts from pathogens, and the like.

[0027] Excessive polymer conjugation and/or conjugation involving atherapeutic moietie's active site where groups associated withbioactivity are found, however, often result in loss of activity and,thus, therapeutic usefulness. This is often the case with lowermolecular weight peptides which have few attachment sites not associatedwith bioactivity. For example, Benhar et al. (Bioconjugate Chem.5:321-326 (1994)) observed that PEGylation of a recombinant single-chainimmunotoxin resulted in the loss of specific target immunoreactivity ofthe immunotoxin. The loss of activity of the immunotoxin was the resultof PEG conjugation at two lysine residues within the antibody-combiningregion of the immunotoxin. To overcome this problem, Benhar et al.replaced these two lysine residues with arginine residues and were ableto obtain an active immunotoxin that was 3-fold more resistant toinactivation by derivatization.

[0028] Another suggestion for overcoming these problems discussed aboveis to use longer, higher molecular weight polymers. These materials,however, are difficult to prepare and expensive to use. Further, theyprovide little improvement over more readily available polymers.

[0029] Another alternative suggested is to attach two strands of polymervia a triazine ring to amino groups of a protein. See, for example,Enzyme 26:49-53 (1981) and Proc. Soc. Exper. Biol. Med.,188:364-369(1988). However, triazine is a toxic substance that isdifficult to reduce to acceptable levels after conjugation. Thus,non-triazine-based activated polymers would offer substantial benefitsto the art.

SUMMARY OF THE INVENTION

[0030] The present invention relates to polyalkylene oxide/amino acidsequence conjugates and processes for preparing them. Suitable aminoacid sequences are peptides, such as, single chain polypeptides havingbinding affinity for an antigen, for example, those described by Ladneret al. in U.S. Pat. No. 4,946,778 and Huston et al. in U.S. Pat. No.5,091,513.

[0031] More particularly, the present invention relates to aphysiologically active, substantially non-immunogenic polypeptideconjugate containing at least one polyalkylene oxide strand coupled to asingle chain polypeptide having binding affinity for an antigen. Thesingle chain polypeptide includes:

[0032] (a) a first polypeptide comprising the binding portion of thelight chain variable region of an antibody;

[0033] (b) a second polypeptide comprising the binding portion of theheavy chain variable region of an antibody; and

[0034] (c) at least one peptide linker linking said first and secondpolypeptides (a) and (b) into said single chain polypeptide havingbinding affinity for the antigen.

[0035] In another aspect, the present invention relates to a process forpreparing physiologically active, substantially non-immunogenicpolypeptide compositions. The process includes coupling a polyalkyleneoxide to a single chain polypeptide having the attributes describedabove. Preferably, the poly(alkylene oxides) used herein arepoly(ethylene glycols) that have been activated for coupling to thetarget polypeptide.

[0036] The invention is also directed to a single-chain antigen-bindingpolypeptide-polyalkylene oxide conjugate, comprising:

[0037] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0038] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0039] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0040] wherein the single-chain antigen-binding polypeptide-polyalkyleneoxide conjugate has an antigen binding affinity within a range of aboutone-fold to about ten-fold of the antigen binding affinity of thenative, unconjugated form of the single-chain antigen-bindingpolypeptide.

[0041] The invention is also directed to a single-chain antigen-bindingpolypeptide-polyalkylene oxide conjugate, comprising:

[0042] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0043] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0044] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0045] wherein the single-chain antigen-binding polypeptide-polyalkyleneoxide conjugate has an antigen binding affinity within about ten-fold ofthe antigen binding affinity of the native, unconjugated form of thesingle-chain antigen-binding polypeptide.

[0046] The invention is also directed to a single-chain antigen-bindingpolypeptide-polyalkylene oxide conjugate, comprising:

[0047] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0048] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0049] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0050] wherein the single-chain antigen-binding polypeptide-polyalkyleneoxide conjugate has an antigen binding affinity within about five-foldof the antigen binding affinity of the native, unconjugated form of thesingle-chain antigen-binding polypeptide.

[0051] The invention is also directed to a single-chain antigen-bindingpolypeptide-polyalkylene oxide conjugate, comprising:

[0052] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0053] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0054] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0055] wherein the single-chain antigen-binding polypeptide-polyalkyleneoxide conjugate has an antigen binding affinity within about two-fold ofthe antigen binding affinity of the native, unconjugated form of thesingle-chain antigen-binding polypeptide.

[0056] The invention is also directed to a single-chain antigen-bindingpolypeptide capable of polyalkylene oxide conjugation, comprising:

[0057] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0058] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0059] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0060] wherein the single-chain antigen-binding polypeptide has at leastone Cys residue wherein the Cys residue is capable of polyalkylene oxideconjugation and the Cys residue is located at a position selected fromthe group consisting of (i) the amino acid position 11, 12, 13, 14 or 15of the light chain variable region; (ii) the amino acid position 77, 78or 79 of the light chain variable region; (iii) the amino acid position11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the aminoacid position 82B, 82C or 83 of the heavy chain variable region; (v) anyamino acid position of the peptide linker; (vi) adjacent to theC-terminus of polypeptide (a) or (b); and (vii) combinations thereof,wherein the polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is capable of binding an antigen.

[0061] The invention is also directed to a single-chain antigen-bindingpolypeptide capable of polyalkylene oxide conjugation, comprising:

[0062] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0063] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0064] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0065] wherein the single-chain antigen-binding polypeptide has at leastthree consecutive Lys residues wherein the consecutive Lys residues arecapable of polyalkylene oxide conjugation and any one of the consecutiveLys residues is located at a position selected from the group consistingof (i) any amino acid position of the peptide linker; (ii) adjacent tothe C-terminus of polypeptide (a) or (b); and (iii) combinationsthereof, wherein the polyalkylene oxide conjugated single-chainantigen-binding polypeptide is capable of binding an antigen. Theseconsecutive lysine residues in the sFv (SCA) protein (i.e., oligo-lysinesFv) generate a “hot spot” for polyalkylene oxide conjugation.

[0066] The invention is also directed to a single-chain antigen-bindingpolypeptide capable of polyalkylene oxide conjugation, comprising:

[0067] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0068] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0069] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0070] wherein the single-chain antigen-binding polypeptide has at leasttwo consecutive Cys residue wherein the consecutive Cys residues arecapable of polyalkylene oxide conjugation and any one of the consecutiveCys residues is located at a position selected from the group consistingof (i) any amino acid position of the peptide linker; (ii) adjacent tothe C-terminus of polypeptide (a) or (b); and (iii) combinationsthereof, wherein the polyalkylene oxide conjugated single-chainantigen-binding polypeptide is capable of binding an antigen. Theseconsecutive cysteine residues in the sFv (SCA) protein (i.e.,oligo-cysteine sFv) generate a “hot spot” for polyalkylene oxideconjugation.

[0071] The invention is further directed to a genetic sequence encodinga single-chain antigen-binding polypeptide capable of polyalkylene oxideconjugation, comprising:

[0072] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0073] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0074] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0075] wherein the single-chain antigen-binding polypeptide has at leastone Cys residue wherein the Cys residue is capable of polyalkylene oxideconjugation and the Cys residue is located at a position selected fromthe group consisting of (i) the amino acid position 11, 12, 13, 14 or 15of the light chain variable region; (ii) the amino acid position 77, 78or 79 of the light chain variable region; (iii) the amino acid position11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the aminoacid position 82B, 82C or 83 of the heavy chain variable region; (v) anyamino acid position of the peptide linker; (vi) adjacent to theC-terminus of polypeptide (a) or (b); and (vii) combinations thereof,wherein the polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is capable of binding an antigen.

[0076] The invention is further directed to a genetic sequence encodinga single-chain antigen-binding polypeptide capable of polyalkylene oxideconjugation, comprising:

[0077] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0078] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0079] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0080] wherein the single-chain antigen-binding polypeptide has at leastthree consecutive Lys residue wherein the consecutive Lys residues arecapable of polyalkylene oxide conjugation and any one of the consecutiveLys residues is located at a position selected from the group consistingof (i) any amino acid position of the peptide linker; (ii) adjacent tothe C-terminus of polypeptide (a) or (b); and (iii) combinationsthereof, wherein the polyalkylene oxide conjugated single-chainantigen-binding polypeptide is capable of binding an antigen. Theseconsecutive lysine residues in the sFv (SCA) protein (i.e., oligo-lysinesFv) generate a “hot spot” for polyalkylene oxide conjugation.

[0081] The invention is further directed to a genetic sequence encodinga single-chain antigen-binding polypeptide capable of polyalkylene oxideconjugation, comprising:

[0082] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0083] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0084] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0085] wherein the single-chain antigen-binding polypeptide has at leasttwo consecutive Cys residue wherein the consecutive Cys residues arecapable of polyalkylene oxide conjugation and any one of the consecutiveCys residues is located at a position selected from the group consistingof (i) any amino acid position of the peptide linker; (ii) adjacent tothe C-terminus of polypeptide (a) or (b); and (iii) combinationsthereof, wherein the polyalkylene oxide conjugated single-chainantigen-binding polypeptide is capable of binding an antigen. Theseconsecutive cysteine residues in the sFv (SCA) protein (i.e.,oligo-cysteine sFv) generate a “hot spot” for polyalkylene oxideconjugation.

[0086] The genetic sequence may be DNA or RNA.

[0087] The invention is directed to a replicable cloning or expressionvehicle comprising the above described DNA sequence. The invention isalso directed to such vehicle which is a plasmid. The invention isfurther directed to a host cell transformed with the above describedDNA. The host cell may be a bacterial cell, a yeast cell or other fungalcell, an insect cell or a mammalian cell line. A preferred host isPichia pastoris.

[0088] The invention is directed to a method of producing a single-chainantigen-binding polypeptide capable of polyalkylene oxide conjugation,comprising:

[0089] (a) providing a first genetic sequence encoding a firstpolypeptide comprising the antigen binding portion of the variableregion of an antibody heavy or light chain;

[0090] (b) providing a second genetic sequence encoding a secondpolypeptide comprising the antigen binding portion of the variableregion of an antibody heavy or light chain; and

[0091] (c) linking the first and second genetic sequences (a) and (b)with a third genetic sequence encoding a peptide linker into a fourthgenetic sequence encoding a single chain polypeptide having an antigenbinding site,

[0092] wherein the single-chain antigen-binding polypeptide has at leastone Cys residue wherein the Cys residue is capable of polyalkylene oxideconjugation and the Cys residue is located at a position selected fromthe group consisting of(i) the amino acid position 11, 12, 13, 14 or 15of the light chain variable region; (ii) the amino acid position 77, 78or 79 of the light chain variable region; (iii) the amino acid position11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the aminoacid position 82B, 82C or 83 of the heavy chain variable region; (v) anyamino acid position of the peptide linker; (vi) adjacent to theC-terminus of polypeptide (a) or (b); and (vii) combinations thereof,wherein the polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is capable of binding an antigen;

[0093] (d) transforming a host cell with the fourth genetic sequenceencoding a single-chain antigen-binding polypeptide of (c); and

[0094] (e) expressing the single-chain antigen-binding polypeptide of(c) in the host, thereby producing a single-chain antigen-bindingpolypeptide capable of polyalkylene oxide conjugation.

[0095] The invention is further directed to a multivalent single-chainantigen-binding protein, comprising two or more single-chainantigen-binding polypeptides, each single-chain antigen-bindingpolypeptide comprising:

[0096] (a) a first polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain;

[0097] (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and

[0098] (c) a peptide linker linking the first and second polypeptides(a) and (b) into a single chain polypeptide having an antigen bindingsite,

[0099] wherein the single-chain antigen-binding polypeptide has at leastone Cys residue wherein the Cys residue is capable of polyalkylene oxideconjugation and the Cys residue is located at a position selected fromthe group consisting of (i) the amino acid position 11, 12, 13, 14 or 15of the light chain variable region; (ii) the amino acid position 77, 78or 79 of the light chain variable region; (iii) the amino acid position11, 12, 13, 14 or 15 of the heavy chain variable region; (iv) the aminoacid position 82B, 82C or 83 of the heavy chain variable region; (v) anyamino acid position of the peptide linker; (vi) adjacent to theC-terminus of polypeptide (a) or (b); and (vii) combinations thereof,wherein the polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is capable of binding an antigen.

[0100] In the above described embodiments of the invention, the Cyspolyalkylene oxide conjugation sequence may be capable of attaching apolyalkylene oxide moiety and the Cys residue is located at a positionselected from the group consisting of (i′) the amino acid position 77 ofthe light chain variable region; (ii′) the amino acid position 82B ofthe heavy chain variable region; (iii′) the amino acid position 3 of thepeptide linker; (iv′) adjacent to the C-terminus of polypeptide (a) or(b); (v′) N-terminus and C-terminus; and (vi′) combinations thereof,wherein the polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is capable of binding an antigen.

[0101] In the above described embodiments of the invention, theoligo-Lys polyalkylene oxide conjugation sequence may be capable ofattaching a polyalkylene oxide moiety at the oligo-Lys residues locatedadjacent to the C-terminus of the protein, wherein the polyalkyleneoxide conjugated single-chain antigen-binding polypeptide is capable ofbinding an antigen.

[0102] In the above described embodiments of the invention, theC-terminus of the second polypeptide (b) may be the native C-terminus.The C-terminus of the second polypeptide (b) may comprise a deletion ofone or plurality of amino acid residue(s), such that the remainingN-terminus amino acid residues of the second polypeptide are sufficientfor the polyalkylene oxide conjugated polypeptide to be capable ofbinding an antigen. The C-terminus of the second polypeptide maycomprise an addition of one or plurality of amino acid residue(s), suchthat the polyalkylene oxide conjugated polypeptide is capable of bindingan antigen.

[0103] In a preferred embodiment of the invention, the first polypeptide(a) may comprise the antigen binding portion of the variable region ofan antibody light chain and the second polypeptide (b) comprises theantigen binding portion of the variable region of an antibody heavychain.

[0104] The invention is also directed to a method of detecting anantigen suspected of being in a sample, comprising:

[0105] (a) contacting the sample with the polyalkylene oxide conjugatedpolypeptide or protein of the invention, wherein the polyalkylene oxideconjugated polypeptide is conjugated to one or plurality of detectablelabel molecule(s), or conjugated to a carrier having one or plurality ofdetectable label molecule(s) bound to the carrier; and

[0106] (b) detecting whether the polyalkylene oxide conjugatedsingle-chain antigen-binding polypeptide has bound to the antigen.

[0107] The invention is further directed to a method of imaging theinternal structure of an animal, comprising administering to the animalan effective amount of the polyalkylene oxide conjugated polypeptide orprotein of the invention, wherein the polyalkylene oxide conjugatedpolypeptide is conjugated to one or plurality of detectable label orchelator molecule(s), or conjugated to a carrier having one or pluralityof detectable label or chelator molecule(s) bound to the carrier, andmeasuring detectable radiation associated with the animal. Animalincludes human and nonhuman.

[0108] The invention is also directed to a method for treating atargeted disease, comprising administering an effective amount of acomposition comprising the polyalkylene oxide conjugated polypeptide orprotein of the invention and a pharmaceutically acceptable carriervehicle, wherein the polyalkylene oxide conjugated polypeptide isconjugated to one or plurality of bioactive molecules, such as peptides,lipids, nucleic acids (i.e., phosphate-lysine complexes), drug, toxin,boron addend or radioisotope molecule(s), or conjugated to a carrierhaving one or plurality of peptides, lipids, nucleic acids (i.e.,phosphate-lysine complexes), drug, toxin, boron addend or radioisotopemolecule(s) bound to the carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0109]FIG. 1 is a graphical representation of three competition ELISA'sin which unlabeled PEG modified CC49/212 SCA (closed squares), CC49/212SCA (open squares), CC49 IgG (open circles), and MOPC-21 IgG (+)competed against a CC49 IgG radiolabeled with ¹²⁵I for binding to theTAG-72 antigen on a human breast carcinoma extract.

[0110]FIG. 2 shows the DNA and protein sequence of CC49/218 SCA whichhas four engineered cysteine residues at the positions indicated by thecodons underlined and marked by an asterisk. Also highlighed are the CDRsequences (double underlined) and the 218 linker (underlined andlabeled). In addition, there are four natural cysteine residues in theprotein which are involved in two disulfide bonds. These are notunderlined. The four engineered cysteine residues occur independently infour different mutants currently, but may be combined in the exactfour-mutant codon version shown in this figure.

[0111]FIG. 3 shows the DNA and protein sequence of CC49/218 SCA with anengineered oligo-lysine C-terminal tail segment. The eight new lysineresidues were genetically engineered at a BstEII site and are shownunderlined and marked with asterisks. Also highlighted are the CDRsequences (double underlined), the 218 linker (underlined and labeled)and selected restriction sites.

[0112]FIG. 4 is a graphical representation of three competition ELISA'sin which unlabeled SC-PEG unreacted CC49/218 SCA (closed squares),CC49/218 SCA (open squares), unlabeled XUS-PEG unreacted CC49/218 SCA(open circles), SC-PEG modified CC49/218 SCA (closed circles), XUS-PEGmodified CC49/218 SCA (open triangles), CC49 IgG (closed triangles), anAnti-FITC SCA (dashed line) or BL-3 IgG (dotted line) were competedagainst a CC49 IgG radiolabeled with ¹²⁵I for binding to the TAG-72antigen on a human breast carcinoma extract.

[0113]FIG. 5 shows the pharmacokinetics of plasma retention of SCA andPEG-SCA. The details of the experiment are described in Example 13.

[0114]FIG. 6 shows an SDS-PAGE of the purified CC49-multimerscross-linked by PEG5000 under reducing conditions. The details of theexperiment are described in Example 14. The lanes of the gel contain thefollowing: 1) trimeric form; 2) dimeric form; 3) dimeric form; 4) mixedpopulation; 5) native CC49; 6) PEG-CC49 monomer; 7) PEG-CC49 monomer; 8)empty; 9) empty; and 10) molecular weight standards.

[0115]FIG. 7 shows the binding kinetics of Mono-, Di-, Tri-, -PEG-CC49.The details of the experiment are described in Example 14. Native CC49is represented by the solid box. PEG-mono-CC49 is represented by theopen box. PEG-Di-CC49 is represented by the solid diamond. PEG-Tri-CC49is represented by the open diamond.

[0116]FIG. 8 shows the results of the competition assay performed inExample 16. Nat is native CC49-SCA, C2 is PEG SC2000-CC49-SCA; GC isglyco-CC49-SCA; B* is the biotinylated CC49-SCA; C12 is thePEG-SC12,000-CC49-SCA; F5 is PEG-Flan-5000-CC49-SCA; and C20 isPEG-SC20000-CC49-SCA.

DESCRIPTION OF THE EMBODIMENTS

[0117] The present invention is directed to the novel combination of apolyalkylene glycol and a single chain polypeptide having bindingaffinity for an antigen, the polyalkylene glycol and polypeptidepreferably beingjoined together by means of a coupling agent.

[0118] Single Chain Polypeptides

[0119] The invention relates to the discovery that polyalkylene oxideconjugated single-chain antigen-binding proteins (“SCA”) or single-chainvariable fragments of antibodies (“sFv”), such as PEGylated SCAproteins, have significant utility beyond that of the nonPEGylatedsingle-chain antigen-binding proteins. In addition to maintaining anantigen binding site, a PEGylated SCA protein has a PEG moiety whichreduces antigenicity and increases the half life of the modifiedpolypeptide in the bloodstream. Accordingly, the invention is directedto monovalent and multivalent SCA proteins capable of PEGylation,compositions of monovalent and multivalent PEGylated SCA proteins,methods of making and purifying monovalent and multivalent PEGylated SCAproteins, and uses for PEGylated SCA proteins. The invention is alsodirected to PEGylated SCA proteins having a diagnostic or therapeuticagent covalently attached to an Cys-linked PEGylated polypeptide or anoligo-Lys linked PEGylated polypeptide.

[0120] The terms “single-chain antigen-binding molecule” (SCA) or“single-chain Fv” (sFv) are used interchangeably. They are structurallydefined as comprising the binding portion of a first polypeptide fromthe variable region of an antibody V_(L) (or V_(H)), associated with thebinding portion of a second polypeptide from the variable region of anantibody V_(H) (or V_(L)), the two polypeptides being joined by apeptide linker linking the first and second polypeptides into a singlepolypeptide chain, such that the first polypeptide is N-terminal to thelinker and second polypeptide is C-terminal to the first polypeptide andlinker. The single polypeptide chain thus comprises a pair of variableregions connected by a polypeptide linker. The regions may associate toform a functional antigen-binding site, as in the case wherein theregions comprise a light-chain and a heavy-chain variable region pairwith appropriately paired complementarity determining regions (CDRs). Inthis case, the single-chain protein is referred to as a “single-chainantigen-binding protein” or “single-chain antigen-binding molecule.”

[0121] Single-chain Fvs can and have been constructed in several ways.Either V_(L) is the N-terminal domain followed by the linker and V_(H)(a V_(L)-Linker-V_(H) construction) or V_(H) is the N-terminal domainfollowed by the linker and V_(L) (V_(H)-Linker-V_(L) construction). Thepreferred embodiment contains V_(L) in the N-terminal domain (see,Anand, N. N., et al., J. Biol. Chem. 266:21874-21879 (1991)).Alternatively, multiple linkers have also been used. Several types ofsFv (SCA) proteins have been successfully constructed and purified, andhave shown binding affinities and specificities similar to theantibodies from which they were derived.

[0122] A description of the theory and production of single-chainantigen-binding proteins is found in Ladner et al., U.S. Pat. Nos.4,946,778, 5,260,203, 5,455,030 and 5,518,889, and in Huston et al.,U.S. Pat. No. 5,091,513 (“biosynthetic antibody binding sites” (BABS)),all incorporated herein by reference. The single-chain antigen-bindingproteins produced under the process recited in the above patents havebinding specificity and affinity substantially similar to that of thecorresponding Fab fragment.

[0123] Typically, the Fv domains have been selected from the group ofmonoclonal antibodies known by their abbreviations in the literature as26-10, MOPC 315, 741F8, 520C9, McPC 603, D1.3, murine phOx, human phOx,RFL3.8 sTCR, 1A6, Se155-4,18-2-3,4-4-20,7A4-1, B6.2, CC49,3C2,2c,MA-15C5/K₁₂GO, Ox, etc. (see, Huston, J. S. et al., Proc. Natl. Acad.Sci. USA 85:5879-5883 (1988); Huston, J. S. et al., SIM News 38(4)(Supp):11 (1988); McCartney, J. et al., ICSU Short Reports 10:114(1990); McCartney, J. E. et al., unpublished results (1990); Nedelman,M. A. et al., J. Nuclear Med. 32 (Supp.):1005 (1991); Huston, J. S. etal., In: Molecular Design and Modeling: Concepts and Applications, PartB, edited by J. J. Langone, Methods in Enzymology 203:46-88 (1991);Huston, J. S. et al., In: Advances in the Applications of MonoclonalAntibodies in Clinical Oncology, Epenetos, A. A. (Ed.), London, Chapman& Hall (1993); Bird, R. E. et al., Science 242:423-426 (1988); Bedzyk,W. D. et al., J. Biol. Chem. 265:18615-18620 (1990); Colcher, D. et al.,J. Nat. Cancer Inst. 82:1191-1197 (1990); Gibbs, R. A. et al., Proc.Natl. Acad. Sci. USA 88:4001-4004 (1991); Milenic, D. E. et al., CancerResearch 51:6363-6371 (1991); Pantoliano, M. W. et al., Biochemistry30:10117-10125 (1991); Chaudhary, V. K. et al., Nature 339:394-397(1989); Chaudhary, V. K. et al., Proc. Natl. Acad. Sci. USA 87:1066-1070(1990); Batra, J. K. et al., Biochem. Biophys. Res. Comm. 171:1-6(1990); Batra, J. K. et al., J. Biol. Chem. 265:15198-15202 (1990);Chaudhary, V. K. et al., Proc. Natl. Acad Sci. USA 87:9491-9494 (1990);Batra, J. K. et al., Mol. Cell. Biol. 11:2200-2205 (1991); Brinkmann, U.et al., Proc. Natl. Acad. Sci. USA 88:8616-8620 (1991); Seetharam, S. etal., J. Biol. Chem. 266:17376-17381 (1991); Brinkmann, U. et al., Proc.Natl. Acad. Sci. USA 89:3075-3079 (1992); Glockshuber, R. et al.,Biochemistry 29:1362-1367 (1990); Skerra, A. et al., Bio/Technol.9:273-278 (1991); Pack, P. et al., Biochemistry 31:1579-1534 (1992);Clackson, T. et al., Nature 352:624-628 (1991); Marks, J. D. et al., J.Mol. Biol. 222:581-597 (1991); Iverson, B. L. et al., Science249:659-662 (1990); Roberts, V. A. et al., Proc. Natl. Acad. Sci. USA87:6654-6658 (1990); Condra, J. H. et al., J. Biol. Chem. 265:2292-2295(1990); Laroche, Y. et al., J. Biol. Chem. 266:16343-16349 (1991);Holvoet, P. et al., J. Biol. Chem. 266:19717-19724 (1991); Anand, N. N.et al., J. Biol. Chem. 266:21874-21879 (1991); Fuchs, P. et al., BiolTechnol. 9:1369-1372 (1991); Breitling, F. et al., Gene 104:104-153(1991); Seehaus, T. et al., Gene 114:235-237 (1992); Takkinen, K. etal., Protein Engng. 4:837-841 (1991); Dreher, M. L. et al., J. Immunol.Methods 139:197-205 (1991); Mottez, E. et al., Eur. J. Immunol.21:467-471 (1991); Traunecker, A. et al., Proc. Natl. Acad. Sci. USA88:8646-8650 (1991); Traunecker, A. et al., EMBO J. 10:3655-3659 (1991);Hoo, W. F. S. et al., Proc. Natl. Acad. Sci. USA 89:4759-4763 (1993)).

[0124] Linkers of the invention used to construct sFv (SCA) polypeptidesare designed to span the C-terminus of V_(L) (or neighboring sitethereof) and the N-terminus of V_(H) (or neighboring site thereof) orbetween the C-terminus of V_(H) and the N-terminus of V_(L). Thepreferred length of the peptide linker should be from 2 to about 50amino acids. In each particular case, the preferred length will dependupon the nature of the polypeptides to be linked and the desiredactivity of the linked fusion polypeptide resulting from the linkage.Generally, the linker should be long enough to allow the resultinglinked fusion polypeptide to properly fold into a conformation providingthe desired biological activity. Where conformational information isavailable, as is the case with sFv (SCA) polypeptides discussed below,the appropriate linker length may be estimated by consideration of the3-dimensional conformation of the substituent polypeptides and thedesired conformation of the resulting linked fusion polypeptide. Wheresuch information is not available, the appropriate linker length may beempirically determined by testing a series of linked fusion polypeptideswith linkers ofvarying lengths for the desired biological activity. Suchlinkers are described in detail in WO 94/12520, incorporated herein byreference.

[0125] Preferred linkers used to construct sFv (SCA) polypeptides havebetween 10 and 30 amino acid residues. The linkers are designed to beflexible, and it is recommended that an underlying sequence ofalternating Gly and Ser residues be used. To enhance the solubility ofthe linker and its associated single chain Fv protein, three chargedresidues may be included, two positively charged lysine residues (K) andone negatively charged glutamic acid residue (E). Preferably, one of thelysine residues is placed close to the N-terminus of V_(H), to replacethe positive charge lost when forming the peptide bond of the linker andthe V_(H). Such linkers are described in detail in U.S. patentapplication Ser. No. 08/224,591, filed Apr. 7, 1994, incorporated hereinby reference. See also, Whitlow, M., et al., Protein Engng.7:1017-1026(1994).

[0126] For multivalent sFvs (SCA), the association of two or more sFvs(SCA) is required for their formation. Although, multivalent sFvs (SCA)can be produced from sFvs (SCA) with linkers as long as 25 residues,they tend to be unstable. Holliger, P., et al., Proc. Natl. Acad. Sci.USA 90:6444-6448 (1993), have recently demonstrated that linkers 0 to 15residues in length facilitate the formation of divalent Fvs. See,Whitlow, M., et al., Protein Engng. 7:1017-1026 (1994); Hoogenboom, H.R., Nature Biotech. 15:125-126 (1997); and WO 93/11161.

[0127] An object of the present invention is to produce a single-chainantigen-binding polypeptide-polyalkylene oxide conjugate which retainsantigen binding affinity within a range of about two-fold to aboutten-fold of the antigen binding affinity of the native single-chainantigen-binding polypeptide.

[0128] Another object of the present invention is to produce an sFv(SCA) having one or more Cys residues such that the Cys residue iscapable of being conjugated with PEG and the PEGylated polypeptide iscapable of binding an antigen (i.e., the PEGylated polypeptide's abilityto bind an antigen is not disrupted). A further object of the presentinvention is to produce an sFv (SCA) having three or more consecutiveLys residues such that the Lys residues are capable of being conjugatedwith PEG and the PEGylated polypeptide is capable of binding an antigen(i.e., the PEGylated polypeptide's ability to bind an antigen is notdisrupted). These novel sFv (SCA) proteins may be conjugated toactivated polyethylene glycol (PEG) such that the PEG modificationoccurs only (or preferentially) at the specifically engineered sites.The activated PEG molecules would be thiol-reactive or amine-reactivepolymers such as are well known in the art. The designed changescorrespond to amino acid residues on the sFv (SCA) surface which arewell separated spatially from the antigen-binding site as deduced fromknown three-dimentional models of the antibody Fv domain.

[0129] A further object of the invention is to produce monovalent andmultivalent sFvs (SCA) having one or more Cys PEG conjugationsequence(s). A further object of the invention is to produce monovalentand multivalent sFvs (SCA) having three or more consecutive Lys (i.e.,oligo-Lys) PEG conjugation sequence(s). For multivalent sFv (SCA), theassociation of two or more sFvs (SCAs) is required for their formation.For example, multivalent sFvs (SCAs) may be generated by chemicallycrosslinking two sFvs (SCAs) with C-terminal cysteine residues (Cumberet al., J. Immunol. 149:120-126 (1992)) and by linking two sFvs (SCAs)with a third polypeptide linker to form a dimeric Fv (SCA)(George etal., J. Cell. Biochem. 15E: 127 (1991)). Details for producingmultivalent sFvs (SCAs) by aggregation are described in Whitlow, M., etal., Protein Engng. 7:1017-1026 (1994). Multivalent antigen-bindingfusion proteins of the invention can be made by any process, butpreferably according to the process for making multivalentantigen-binding proteins set forth in WO 93/11161, incorporated hereinby reference.

[0130] Identification and Synthesis of Site Specific PEGylationSequences

[0131] In the present invention, Cys PEGylation sites may occur in theV_(L) and V_(H) regions, adjacent to the C-terminus of the polypeptide(V_(L), V_(H) or neighboring site thereof), the N-terminus of thepolypeptide (V_(L), V_(H) or neighboring site thereof), the linkerregion between the first and second polypeptide regions, or occur in acombination of these regions. In the present invention, oligo-LysPEGylation sites may occur in the polypeptide linker or in theC-terminus or adjacent to the C-terminus of the polypeptide. The designof the PEG conjugaton sites on a protein involves examining thestructural information known about the protein and the residues in theproteins involved in antigen binding. The PEG conjugaton sites arechosen to be as far from these residues as possible so as to preventdisruption of the antigen-binding site.

[0132] The Cys or the oligo-Lys PEGylation site may occur in (1) thenative C-terminus of V_(L) (or V_(H)), (2) the C-terminus of V_(L) (orV_(H)) wherein the C-terminus has a deletion of one or plurality ofamino acid residue(s), such that the remaining N-terminus amino acidresidues of the peptide are sufficient for the PEGylated polypeptide tobe capable of binding an antigen or (3) the C-terminus of V_(L) (orV_(H)) wherein the C-terminus has an addition of one or plurality ofamino acid residue(s), such that the remaining N-terminus amino acidresidues of the peptide are sufficient for the PEGylated polypeptide tobe capable of binding an antigen. By “native” is intended the naturallyoccurring C-terminus of the immunoglobulin (first or secondpolypeptide). By “C-terminus” it is well understood in the art asintending the C-terminal amino acid residue or the C-terminal region ofthe polypeptide, which could include up to all of the amino acidresidues of the polypeptide excluding the first N-terminal amino acidresidue of the polypeptide. However, in the present invention,“C-terminus” is intended as the C-terminal amino acid residue of theabove mentioned three types of C-terminus (1, 2, or 3), unless otherwiseindicated or intended.

[0133] PEGylation sites were identified and engineered at residueswithin loop sites in regions of the sFv (SCA) that are diametricallyopposed to the antigen binding site. The five loop regions andC-terminal extension chosen as preferred sites of glycosylation areamong the most distant regions spatially removed from the binding site.

[0134] The six furthest portions of an sFv (SCA) from the antigenbinding site are as follows:

[0135] 1) The loop made up of residues 11 to 15 in the light chain;

[0136] 2) The loop made up of residues 77 to 79 in the light chain;

[0137] 3) The N-terminus of the linker;

[0138] 4) The loop made up of residues 11 to 15 in the heavy chain;

[0139] 5) The loop made up of residues 82B, 82C and 83 in the heavychain; and

[0140] 6) The C-terminus of the sFv (or SCA).

[0141] The residues are identified as according to Kabat et al.,Sequences of Proteins of Immunological Interest, 5th ed., U.S. Dept.Health and Human Services, Bethesda, Md. (1991). These possiblePEGylation sites were determined by examining the 4-4-20 mouse Fabstructure (see, Whitlow, M. et al., Protein Engng 8:749-761 (1995),incorporated herein by reference).

[0142] After identifying the loops furthest from the antigen bindingsite, the nucleic and amino acid sequences of each loop are examined forpossible Cys PEGylation sites that may be engineered into the loopregion. The engineered placement of the Cys residue anywhere in thesesix identified regions can generate a preferred site for sFv (SCA)PEGylation. The engineered placement of the oligo-Lys residues in thelinker, the C-terminus of the sFv (SCA) and/or adjacent to theC-terminus of the sFv (SCA) can generate a preferred site for sFvPEGylation.

[0143] The design approach described above has been used for theCC49/218 SCA. FIG. 2 shows the following resulting designs: designedPEGylation site no. 1 in the light chain of the CC49/218 SCA; designedPEGylation site no. 2 in the N-terminal end of the linker in CC49/218SCA; designed PEGylation site no. 3 in the heavy chain of the CC49/218SCA; designed PEGylation site no. 4 at the C-terminus of the CC49/218SCA. FIG. 3 shows the following resulting designs: designed oligo-Lys“hot spot” PEGylation sites at the C-terminus of the CC49/218 SCA. Anycombination of these sites could be used.

[0144] The particular nucleotide sequence which is used to introduce aCys or oligo-Lys PEGylation site into the various positions will dependupon the naturally-occurring nucleotide sequence. The most preferredsites are those in which it takes a minimum number of changes togenerate the PEGylation site. Of course, based on the redundancy of thegenetic code, a particular amino acid may be encoded by multiplenucleotide sequences.

[0145] Site-directed mutagenesis is used to change the native proteinsequence to one that incorporates the Cys residue or oligo-Lys residuesfor PEGylation. The mutant protein gene is placed in an expressionsystem, such as bacterial cells, yeast or other fungal cells, insectcells or mammalian cells. The mutant protein can be purified by standardpurification methods.

[0146] Oligonucleotide-directed mutagenesis methods for generating theCys or oligo-Lys PEGylation sites and related techniques for mutagenesisof cloned DNA are well known in the art. See, Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989); Ausubel et al. (eds.), CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons (1987), bothincorporated herein by reference. A preferred oligonucleotide-directedmutagenesis method for the present invention is according to Ho et al.,Gene 77:51-59 (1989), incorporated herein by reference.

[0147] Hosts and Vectors

[0148] After mutating the nucleotide sequence of the sFv (SCA), themutated DNA can be inserted into a cloning vector for further analysis,such as for confirmation of the DNA sequence. To express the polypeptideencoded by the mutated DNA sequence, the DNA sequence is operably linkedto regulatory sequences controlling transcriptional expression andintroduced into either a prokaryotic or eukaryotic host cell.

[0149] Although sFvs (SCAs) are typically produced by prokaryotic hostcells, eukaryotic host cells are the preferred host cells. Preferredhost cells include yeast or other fungal cells, insect cells ormammalian cells. Standard protein purification methods may be used topurify these mutant proteins. Only minor modification to the nativeprotein's purification scheme may be required.

[0150] Also provided by the invention are DNA molecules such as purifiedgenetic sequences or plasmids or vectors encoding the sFv (SCA) of theinvention that have engineered Cys residues and/or oligo-Lys residuescapable of PEG conjugation. The DNA sequence for the PEGylated sFv (SCA)polypeptide can be chosen so as to optimize production in organisms suchas prokaryotes, yeast or other fungal cells, insect cells or mammaliancells.

[0151] The DNA molecule encoding an sFv (SCA) having Cys residues and/oroligo-Lys residues for PEG conjugation can be operably linked into anexpression vector and introduced into a host cell to enable theexpression of the engineered sFv (SCA) protein by that cell. A DNAsequence encoding an sFv (SCA) having Cys and/or oligo-Lys PEGylationsites may be recombined with vector DNA in accordance with conventionaltechniques. Recombinant hosts as well as methods of using them toproduce single chain proteins of the invention are also provided herein.

[0152] The expression of such sFv (SCA) proteins of the invention can beaccomplished in procaryotic cells. Preferred prokaryotic hosts include,but are not limited to, bacteria such as Neisseria, Mycobacteria,Streptococci, Chlamydia and E. coli.

[0153] Eukaryotic hosts for cloning and expression of such sFv (SCA)proteins of the invention include insect cells, yeast, fingi, andmammalian cells (such as, for example, human or primate cells) either invivo, or in tissue culture. A preferred host for the invention is Pichiapastoris.

[0154] The appropriate DNA molecules, hosts, methods of production,isolation and purification of monovalent, multivalent and fusion formsof proteins, especially sFv (SCA) polypeptides, are thoroughly describedin the prior art, such as, e.g., U.S. Pat. No. 4,946,778, which is fullyincorporated herein by reference.

[0155] The sFv (SCA) encoding sequence having Cys residues and/oroligo-Lys residues for PEG conjugation and an operably linked promotermay be introduced into a recipient prokaryotic or eukaryotic cell eitheras a non-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the desired sFv (SCA) protein may occur through the transientexpression of the introduced sequence. Alternatively, permanentexpression may occur through the integration of the introduced sFv (SCA)sequence into the host chromosome.

[0156] In one embodiment, the sFv (SCA) sequence can be integrated intothe host cell chromosome. Cells which have stably integrated theintroduced DNA into their chromosomes can be selected by alsointroducing one or more markers which allow for selection of host cellswhich contain the sFv (SCA) sequence and marker. The marker maycomplement an auxotrophy in the host (such as his4, leu2, or ura3, whichare common yeast auxotrophic markers), biocide resistance, e.g.,antibiotics, or resistance to heavy metals, such as copper, or the like.The selectable marker gene can either be directly linked to the sFv(SCA) DNA sequence to be expressed, or introduced into the same cell byco-transfection.

[0157] In another embodiment, the introduced sequence will beincorporated into a plasmid vector capable of autonomous replication inthe recipient host cell. Any of a wide variety of vectors may beemployed for this purpose. Factors of importance in selecting aparticular plasmid or viral vector include: the ease with whichrecipient cells that contain the vector may be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to “shuttle” the vector between hostcells of different species.

[0158] Any of a series of yeast vector systems can be utilized. Examplesof such expression vectors include the yeast 2-micron circle, theexpression plasmids YEP13, YCP and YRP, etc., or their derivatives. Suchplasmids are well known in the art (Botstein et al., Miami Wntr. Symp.19:265-274 (1982); Broach, J. R., In: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R.,Cell 28:203-204 (1982)).

[0159] For a mammalian host, several possible vector systems areavailable for expression. One class of vectors utilize DNA elementswhich provide autonomously replicating extra-chromosomal plasmids,derived from animal viruses such as bovine papilloma virus, polyomavirus, adenovirus, or SV40 virus. A second class of vectors relies uponthe integration of the desired gene sequences into the host chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes may be selected by also introducing one or more markerswhich allow selection of host cells which contain the expression vector.The marker may provide for prototrophy to an auxotrophic host, biocideresistance, e.g., antibiotics, or resistance to heavy metals, such ascopper or the like. The selectable marker gene can either be directlylinked to the DNA sequences to be expressed, or introduced into the samecell by co-transformation. Additional elements may also be needed foroptimal synthesis of mRNA. These elements may include splice signals, aswell as transcription promoters, enhancers, and termination signals. ThecDNA expression vectors incorporating such elements include thosedescribed by Okayama, H., Mol. Cell. Biol. 3:280 (1983), and others.

[0160] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Preferred vectors for expression inPichia are pHIL-S 1 (Invitrogen Corp.) and pPIC9 (Invitrogen Corp.).Other suitable vectors will be readily apparent to the skilled artisan.

[0161] Once the vector or DNA sequence containing the constructs hasbeen prepared for expression, the DNA constructs may be introduced ortransformed into an appropriate host. Various techniques may beemployed, such as transformation, transfection, protoplast fusion,calcium phosphate precipitation, electroporation, or other conventionaltechniques. After the cells have been transformed with the recombinantDNA (or RNA) molecule, the cells are grown in media and screened forappropriate activities. Expression of the sequence results in theproduction of the mutant sFv (SCA) for PEG conjugation of the presentinvention.

[0162] Straight Chain Polymers

[0163] The straight chain polyalkylene glycols employed in the practiceof the present invention are of the structural formula

[0164] wherein R is selected from the group consisting of hydrogen,lower alkyl and mixtures thereof, R¹ is selected from the groupconsisting of hydrogen and lower alkyl, and n is a positive integer. By“lower alkyl” is meant an alkyl group having from one to four carbonatoms, i.e., methyl, ethyl, propyl, butyl, and isomers of the foregoing.R is preferably selected from the group consisting of hydrogen, methyl,and mixtures thereof, R¹ is preferably selected from the groupconsisting of hydrogen and methyl, and n is preferably a positiveinteger of 500 or less. R is most preferably hydrogen, R¹ is mostpreferably methyl, and n is most preferably an integer of 7 to 150. Itwill be readily apparent to those skilled in the art that the preferredpoly(alkylene glycols) employed in the practice of the present inventionare poly(ethylene glycol), poly(propylene glycol), mixtures thereof, andcopolymers of poly(ethylene glycol) and poly(propylene glycol), whereinone of the terminal hydroxyl groups of the polymer may be substitutedwith a lower alkyl group. A preferred polyalkylene glycol for use in thepresent invention is poly(ethylene glycol)-hydrazine. The most preferredpolyalkylene glycol for use in the present invention is methoxypoly(ethylene glycol).

[0165] Hereinafter, for convenience, the polyalkylene glycol employed inthe practice of the present invention will be designated PAG, which termis intended to include both compounds wherein R¹ is hydrogen andcompounds wherein R¹ is alkyl. PEG refers to poly(ethylene glycol) andmPEG refers to methoxy poly(ethylene glycol).

[0166] The PAG does not have to be of a particular molecular weight, butit is preferred that the molecular weight be between about 500 and about40,000; more preferably, between about 2,000 and about 20,000. Thechoice of molecular weight of PAG is made based on the nature of theparticular polypeptide employed, for example, the number of amino orother groups available on the polypeptide for modification. Molecularweights of about 10,000 and about 20,000 are most preferred.

[0167] It is well known in the art that PAGs that contain two terminalhydroxyl groups per moiety are capable of crosslinking other polymers,e.g. proteins. Where, as is often the case, crosslinking would be deemedundesirable, such crosslinking can be minimized or prevented by meansknown in the art. For example, Davis et al. in U.S. Pat. No. 4,179,337have pointed out that a preferred means for preventing crosslinking isto preblock one end of the PAG, such as is done in the commerciallyavailable methoxy poly(ethylene glycol).

[0168] The PAGs employed in the practice of the present invention arepreferably coupled to polypeptides by means of suitable coupling agents.A useful review of a number of coupling agents that can be employed inthe practice of the present invention appears in Dreborg et al.,Critical Reviews in Therapeutic Drug Carrier Systems 6(4):315-365(1990), see, especially, pp. 317-320.

[0169] Probably the best known coupling agent for this purpose iscyanuric chloride. Its use has been described in numerous references,see, for example, Abuchowski et al., J. Biol. Chem. 252(11):3578-3581(Jun. 10, 1977).

[0170] Zalipsky et al., Eur. Pol. J. 19(12): 1177-1183 (1983), amongothers, have described the reaction of methoxy poly(ethylene glycol)with succinic anhydride:

[0171] It is also known to alkylate mPEG with ethylbromoacetate in thepresence of a base such as K-tertiary butoxide in tertiary butanol,Na-naphthalene in tetrahydrofuran, or butyl lithium in benzene:

[0172] The terminal hydroxyl groups of PEG can be transformed intoamine, carboxyl, or hexamethyl isocyanate groups. See, for example,Zalipsky et al., 1983, supra. A mixed anhydride derivative ofcarboxylated mPEG can be prepared in the presence of triethylamine andthen reacted with proteins:

[0173] Carboxylated mPEG can also be reacted with hydroxysuccimide inthe presence of dicyclohexylcarbodiimide and dimethyl formamide forreaction with protein:

[0174] King and Weiner (Int. J. Peptide Protein Res. 16:147 (1980)describe the dithiocarbonate of mPEG:

[0175] Beauchamp et al., Analytical Biochem. 131:25-33 (1983) describethe activation of PEG with 1,1′-carbonyldiimidazole. Reaction of thisderivative with a peptide yields a carbamate linkage:

[0176] Veronese et al., Appl. Biochem. & Biotechnol. 11:141-152 (1985)describe the activation of methoxy poly(ethylene glycol) withphenylchloroformates, e.g., 2,4,5-trichlorophenylchloroformate orp-nitrophenylchloroformate. These derivatives are linked to peptides byurethane linkages:

[0177] Ueno et al. in European Patent Application 87103259.5 form mPEGimidoesters from the corresponding nitriles by reaction with dryhydrogen chloride in the presence of a dehydrated lower alcohol:

[0178] Abuchowski et al., Cancer Biochem. Biophys. 7:175-186 (1984) havedescribed forming mPEG succinate as described above and then formingmethoxy polyethylene glycolyl succinimidyl succinate (“SS-PEG”) byreaction with hydroxysuccinimide in the presence ofdicyclohexylcarbodiimide:

[0179] Sano et al., European Patent Application No. 89107960.0 disclosethe phenyl glyoxal derivative of methoxy poly(ethylene glycol), which iscapable of modifying the guanidino groups in peptides:

[0180] Zalipsky, in U.S. Pat. No. 5,122,614, describes the activation ofPEG by conversion into its N-succinimide carbonate derivative(“SC-PEG”):

methoxypoly(ethylene glycol)-succinyl carbonate SC-PEG

[0181] Zalipsky et al., J. Macromol. Sci. Chem. A21:839, disclose theamino acid ester derivative of methoxy poly(ethylene glycol):

[0182] Davis et al., U.S. Pat. No. 4,179,337, disclose a hydrazidederivative of methoxy poly(ethylene glycol), which is capable ofmodifying aldehydes and ketones and other functional groups:

[0183] It is further disclosed that the bifunctional derivative of PEG,i.e., polyethylene glycol-bis-succinidyl carbonate (“BSC-PEG”) can beprepared by similar means. The SC-PEG and BSC-PEG compounds are thenreacted with amine groups in a protein and attached thereto via urethane(carbamate) linkages.

[0184] It will be readily apparent to those skilled in the art thatother activated PAGs can also be employed in the practice of the presentinvention. The preferred activated PAG for use in the practice of thepresent invention is selected from the group consisting of SS-PEG andSC-PEG. The use of SC-PEG is most preferred.

[0185] Branched Polymers

[0186] The invention further provides for the use of branched,substantially non-antigenic polymers for polyalkylene oxide conjugationof the sFv (SCA) proteins corresponding to the formula:

(R)_(n)L—A  (II)

[0187] wherein (R) includes a water-soluble non-antigenic polymer;

[0188] (n)=2 or 3;

[0189] (L) is an aliphatic linking moiety covalently linked to each (R);and

[0190] (A) represents an activated functional group capable ofundergoing nucleophilic substitution. For example, (A) can be a groupwhich is capable of bonding with biologically active nucleophiles ormoieties capable of doing the same.

[0191] In particularly preferred aspects of the invention (R) includes apoly(alkylene oxide) PAO such as poly(ethylene glycol) PEG or MPEG. Itis preferred that each chain have a molecular weight of between about200 and about 12,000 daltons and preferably between about 1,000 andabout 10,000 daltons. Molecular weights of about 5,000 daltons are mostpreferred.

[0192] As shown in Formula II, 2 or 3 polymer chains, designated (R)herein, are joined to the alphatic linking moiety (L). Suitablealiphatics included substituted alkyl diamines and triamines, lysineesters and malonic ester derivatives. The lining moieties are perferablynon-planar, so that the polymer chains are not rigidly fixed. Thelinking moiety (L) is also a means for attaching the mulitple polymerchains or “branches” to (A), the moeity through which the polymerattaches to the sFv (SCA) protein.

[0193] (L) preferably includes a multiply-functionalized alykyl groupcontaining up to 18, and more preferably between 1-10 carbon atoms. Aheteroatom such as nitrogen, oxygen or sulfur may be included within thealkyl chain. The alkyl chain may also be branched at a carbon ornitrogen atom. In another aspect of the invention, (L) is a singlenitrogen atom.

[0194] (L) and (R) are preferably joined by a reaction betweennucleophilic functional groups on both (R) and (L). Each (R) is suitablyfunctionalized to undergo nucleophilic substitution and bond with (L).Such functionalization of polymers is readily apparent to those ofordinary skill in the art.

[0195] A wide variety of linkages are contemplated between (R) and (L).Urethane (carbamate) linkages are preferred. The bond can be formed, forexample, by reacting an amino group such as 1,3-diamino-2-propanol withmethoxypolyethylene glycol succinimidyl carbonate as described in U.S.Pat. No. 5,122,614. Amide linkages, which can be formed by reacting anamino-terminated non-antigenic polymer suchas methoxypolyethyleneglycol-amine (MPEG amine) with an acyl chloride functional group.Examples of other such linkages include ether, amine, urea, and thio andthiol analogs thereof, as well as the thio and thiol analogs of theurethane and amide linkages discussed supra.

[0196] The moiety (A) of Formula II represents groups that “activate”the branched polymers of the present invention forconjugationwithbiologically active materials. (A) can be a moietyselected from:

[0197] 1. Functional groups capable of reacting with an amino group suchas:

[0198] a) carbonates such as the p-nitrophenyl or succinimidyl;

[0199] b) carbonyl imidazole;

[0200] c) azlactones;

[0201] d) cyclic imide thiones; or

[0202] e) isocyanates or isothiocyanates.

[0203] 2. Functional groups capable of reacting with carboxylic acidgroups and reactive with carbonyl groups such as:

[0204] a) primary amines; or

[0205] b) hydrazine and hydrazide functional groups such as the acylhydrazides, carbazates, semicarbamates, thiocarbazates, etc.

[0206] 3. Functional groups capable of reacting with mercapto orsulfhydryl groups such as phenyl glyoxals; see, for example, U.S. Pat.No. 5,093,531.

[0207] 4. Other nucleophiles capable of reacting with an electrophiliccenter. A non-limiting list includes, for example, hydroxyl, amino,carboxyl, thiol groups, active methylene and the like.

[0208] The moiety (A) can also include a spacer moiety located proximalto the aliphatic linking moiety (L). The spacer moiety may be aheteroalkyl, alkoxyl, alkyl containing up to 18 carbon atoms or even anadditional polymer chain. The spacer moieties can be added usingstandard synthesis techniques.

[0209] The branched polymers, generally, U-PAO's or U-PEG's, are formedusing conventional reaction techniques known to those of ordinary skillin the art.

[0210] These umbrella-like branched polymers of the present invention(U-PAO's or U-PEG's) react with biologically active nucleophiles to formconjugates. The point of polymer attachment depends upon the functionalgroup (A). For example, (A) can be a succinimidyl succinate or carbonateand react with E-amino lysines. The branched polymers can also beactivated to link with any primary or secondary amino group, mercaptogroup, carboxylic acid group, reactive carbonyl group or the like foundon biologically active polypeptides. Other groups are apparent to thoseof ordinary skill in the art.

[0211] One of the main advantages of the use of the branched polymers isthat the branching imparts an umbrella-like three dimensional protectivecovering to the materials they are conjugated with. This contrasts withthe string-like structure of the straight chain polymers discussed,supra. An additional advantage of the branched ploymers is that theyprovide the benefits associated with attaching several strands ofpolymers to a sFv protein but require substantially fewer conjugationsites. The desired properties of PEGylation are realized and the loss ofbioactivity is minimized.

[0212] One or more of the activated branched polymers can be attached toa biologically active nucleophile, such as an sFv protein, by standardchemical reactions. The conjugate is represented by the formula:

[(R)_(n)L—A¹]_(z)—(nucleophile)  (III)

[0213] wherein (R) is a water-soluble substantially non-antigenicpolymer; n=2 or 3; (L) is an aliphatic linking moiety; (A¹) represents alinkage between (L) and the nucleophile and (z) is an integer≧1representing the number of polymers conjugated to the biologicallyactive nucleophile. The upper limit for (z) will be determined by thenumber of available nucleophilic attachment sites and the degree ofpolymer attachment sought by the artisan. The degree of conjugation canbe modified by varying the reaction stoichimetry using well-knowntechniques. More than one polymer conjugated to the nucleophile can beobtained by reacting a stoichimetric excess of the activated polymerwith the nucleophile.

[0214] Purification of sFv Proteins

[0215] A generic protocol that has been developed and used to producetwelve different single chain antigen binding molecules. It involvescell lysis and washing, solubilization in a denaturing solvent,refolding by dilution, and two ion-exchange HPLC chromatography steps.Such isolated sFvs (SCAs) are capable of being PAG conjugated accordingto the present invention.

[0216] The fermentation of the sFv-producing E. coli strains areperformed at 32° C. using a casein digest-glucose-salts medium. At anoptical density of 18 to 20 at 600 nm, sFv expression is induced by a42° C. temperature shock for one hour. After the fermentation is cooledto 10° C., the cells are harvested by centrifugation at 7000 g for tenminutes. The wet cell paste is then stored frozen at −20° C.Approximately 200 to 300 g of wet cell paste is normally recovered fromone 10-liter fermentation.

[0217] For protein recovery, the cell paste from three 10-literfermentations (600-900 g) is thawed overnight at 4° C. and gentlyresuspended at 4° C. in 50 mM Tris-HCl, 1.0 mM EDTA, 100 mM KCl, 0.1 mMphenylmethylsulfonyl chloride (PMSF), pH 8.0 (lysis buffer), using 10liters of lysis buffer for every kilogram of wet cell paste. Whenthoroughly resuspended, the chilled mixture is passed three timesthrough a Manton-Gaulin cell homogenizer to fully lyse the cells.Because the cell homogenizer raises the temperature of the cell lysateto 25±5° C., the cell lysate is cooled to 5±2° C. with a Lauda/Brinkmanchilling coil after each pass. Complete lysis is verified by visualinspection under a microscope.

[0218] The cell lysate is centrifuged at 24,300 g for thirty minutes at6° C. using a Sorvall RC-5B centrifuge. The pellet contains theinsoluble sFv and the supernatant is discarded. The pellet is washed bygently scraping it from the centrifuge bottles and resuspending it in 5liters of lysis buffer/kg of wet cell paste. The resulting 3.0-4.5-litersuspension is again centrifuged at 24,300 g for 30 min at 6° C., and thesupernatant is discarded. This washing of the cell pellet removessoluble E. coli proteins and can be repeated as many as five times. Atany time during this washing procedure the material can be stored as afrozen pellet at −20° C. A substantial time saving in the washing stepscan be accomplished by utilizing a Pellicon tangential flow apparatusequipped with 0.22-μm microporous filters.

[0219] The washed cell pellet is solubilized at 4° C. in freshlyprepared 6 M guanidine hydrochloride, 50 mM Tris-HCl, 10 mM CaCl₂, 50 mMKCl, pH 8.0 (denaturing buffer), using 6 ml/g of pellet. If necessary, afew quick pulses from a Heat Systems Ultrasonics tissue homogenizer canbe used to complete the solubilization. The resulting suspension iscentrifuged at 24,300 g for 45 minutes at 6° C. and the pellet isdiscarded. The optical density of the supernatant is determined at 280nm and if the OD₂₈₀ is above 30, additional denaturing buffer is addedto obtain an OD₂₈₀ of approximately 25.

[0220] The supernatant is slowly diluted into cold (4-7° C.) refoldingbuffer (50 mM Tris-HCl, 10 mM CaCl₂, 50 mM KCl, 0.1 mM PMSF, pH 8.0)until a 1:10 to 1:100 dilution is reached (final volume 70-120 liters).The refolding buffer should be prepared at least one day prior to use,to allow sufficient time for it to cool to 4° C. The best results willbe obtained when the supernatant is slowly added to the refolding bufferover a two hour period, with gentle mixing. The solution is leftundisturbed for at least twenty hours and then filtered through aMillipore Pellicon tangential flow apparatus at 4° C. with four to six0.45-μm microporous membranes (HVLP 000 C5). The filtrate isconcentrated to 1 to 2 liters using a Pellicon apparatus with four tosix 10,000 NMWL cassettes (SKlPA156A4), again at 4° C.

[0221] The concentrated crude sFv sample is buffer exchanged at 4° C.into 20 mM 2-[N-morpholino]ethanesulfonic acid (Mes), 0.3 mM CaCl₂, pH6.0, using the Pellicon ultrafiltration apparatus equipped with four tosix 10,000 NMWL cassettes. The sample is then chromatographed on aWaters Accell Plus CM ion-exchange (RCM) column (4.7×30.0 cm). Prior toloading on the HPLC, the material is filtered through a 0.22-μm filterand the Accell column is equilibrated with Buffer A (40 mM Mes, 1 mMCaCl₂, pH 6.0). Following sample loading, the Accell column is elutedover a 55-minute period with a linear gradient of Buffer A and Buffer B(40 mM Mes, 100 mM CaCl₂, pH 7.0). (See Table 1). TABLE 1 AccellCation-Exchange HPLC Gradients Buffers^(b) Time (min)^(a) Flow (ml/min)% A % B % C Initial 40.0 100  0  0 55.0 40.0  0 100  0 58.0 40.0  0 100 0 60.0 40.0  0  0 100 62.0 40.0 100  0  0

[0222] The Accell Plus CM column has a capacity of about 3 g and thusall the crude sFv sample can normally be loaded in a single run. Thefractions are analyzed using 4-20% Novex SDS-PAGE gels and the peakfractions are pooled. Normally, the sFv elutes from the Accellion-exchange column quite early in the gradient. To enhance resolutionfor certain sFv proteins, holds in the gradient can be implemented.

[0223] The pooled fractions from the Accell HPLC purification aredialyzed against Buffer D (40 mM 3-[N-morpholino]propanesulfonic acid(Mops), 0.5 mM Ca acetate, pH 6.0) until the conductivity is lowered tothat of Buffer D. The sample is then loaded on a 21.5×150-mmpolyaspartic acid PolyCAT A column. If more than 60 mg is loaded on thiscolumn, the resolution begins to deteriorate; thus, the pooled fractionsfrom the Accell HPLC purification often must be divided into severalPolyCAT A runs. Most sFv proteins have an extinction coefficient ofabout 2.0 mg ml⁻¹ cm¹ at 280 nm and this can be used to determineprotein concentration. The sFv sample is eluted from the PolyCAT Acolumn with a 50-min linear gradient of Buffer D and Buffer E (40 mMMops, 10 mM Ca acetate, pH 8.0). See Table 2. TABLE 2 PoIyCAT ACation-Exchange HPLC Gradients Buffers^(b) Time (min)^(a) Flow (ml/min)% D % E % F Initial 15.0 100  0  0 50.0 15.0  0 100  0 55.0 15.0  0 100 0 60.0 15.0  0  0 100 63.0 15.0  0  0 100 64.0 15.0 100  0  0 67.0 15.0100  0  0

[0224] The sFv proteins will often elute between 20 and 26 min when thisgradient is used. This corresponds to an eluting solvent composition ofapproximately 70% Buffer D and 30% Buffer E.

[0225] This purification procedure yields sFv proteins that are morethan 95% pure as examined by SDS-PAGE and Scatchard analysis.Modifications of the above procedure may be dictated by the isoelectricpoint of the particular sFv being purified, which is often between 8.0and 9.3.

[0226] The polyalkylene glycols (PAGs) employed in the practice of thepresent invention, which, as indicated above, are preferably activatedby reaction with a coupler, can be reacted with any of several groupsthat may be present attached to the chain of the single chain antigenbinding molecules, e.g. terminal carboxyl groups, thiol groups, phenolichydroxyl groups, or primary amino groups located at the chain terminusor along the chain. It is preferred to react activated PAGs with primaryamine groups, especially those occurring along the peptide chain. It ismost preferred that the activated PAGs be coupled to the E amino groupsof lysine residues as well as cysteine residues in the polypeptide.

[0227] The reaction between the PAG and the single chain polypeptide isnormally carried out in solution, preferably an aqueous buffer solutionproviding a pH in the range of from about 6 to about 10, preferably fromabout 7 to about 9, most preferably from about 7 to about 8. As examplesof buffer solutions that will provide pH's in these ranges at 25° C. maybe listed:

[0228] 50 ml of 0.1 molar potassium dihydrogen phosphate +5.6 to 46.1 ml0.1 molar NaOH diluted to 100 ml

[0229] 50 ml of 0.025 molar borate+2.0 to 20.5 ml 0.1 molar HCl dilutedto 100 ml

[0230] 50 ml of 0.025 molar borate+0.9 to 18.3 ml 0.1 molar NaOH dilutedto 100 ml

[0231] 50 ml of 0.05 molar sodium bicarbonate+5.0 to 10.7 ml 0.1 molarNaOH diluted to 100 ml

[0232] The precise adjustment of the quantity of acid or base to be usedto provide a particular desired pH will be readily determinable by thoseskilled in the art.

[0233] If, in a given instance, the use of a biological buffer should berequired, one of the following may be employed:

[0234] 3-(N-Morpholino)propanesulfonic acid (MOPS)

[0235] 3-(N-Morpholino)-2-hydroxypropanesufonic acid (MOPSO)

[0236] Piperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (POPSO)

[0237] The reaction between the PAG and the single chain polypeptidewill normally be run under conditions that will not give rise todenaturation, e.g. mild temperatures and no more agitation thannecessary. The reaction will preferably be run at a temperature in therange of from about 4° C. to about 25° C. More preferably, the reactionwill be run at room temperature, i.e. from about 20° C. to about 25° C.

[0238] It will be readily understood by those skilled in the art thatthe amount of PAG employed relative to the amount of single chainpolypeptide will be dependent upon the desired nature of the reactionproduct. Where, for example, it is desired to react a PAG with eachlysine residue along the polypeptide chain, an amount of PAG at leastequimolar to the lysine concentration will be required. It will beadvantageous to employ an excess of PAG, where possible, in order toincrease the reaction rate and the likelihood of a complete reaction.Clearly, if fewer than all of the possible reaction sites along thepolypeptide chain are to be derivatized, correspondingly less PAG willbe used. In general, however, where molar excesses of PAG's are used, ithas been determined that molar excesses on the order of 2-100 of the PAGcan be used; molar excesses of 2-10 are preferred.

[0239] The time required for the reaction will depend upon a number offactors, such as reaction temperature, concentration of reactants, andwhether full or partial reaction is desired. The course of the reactioncan be monitored by conventional means, such as the analysis of periodicsamples by size exclusion chromatography or gel electrophoresis. Thereaction can conveniently be terminated when desired by the addition ofa compound having a primary amine group, e.g. glycine, to scavenge theexcess PAG. A reaction time of about 15-120 minutes will typically berequired to fully react the PAG with the primary amine groups of thelysine residues of the single chain polypeptide at room temperature. Theskilled practitioner will understand that the time for conjugation, aswell as the amount and type of PAG, must not be such as to inactivatethe polypeptide being employed.

[0240] Purification of the PAG/single chain polypeptide reaction productcan be effected by means commonly employed by those skilled in the art,such as, for example, size exclusion chromatography, ion-exchangechromatography, ultrafiltration, dialysis, and the like. Solutions ofthe reaction product can, if desired, be concentrated with a rotaryevaporator and can be obtained in the dry state by lyophilization.

[0241] Depending upon the particular single chain antigen bindingmolecule chosen and the extent to which it is reacted with the PAG, theresulting adduct is expected to be useful both diagnostically andtherapeutically, exhibiting, as compared to the unreacted single chainpolypeptide, decreased immunogenicity, increased circulating life, andincreased stability while maintaining an acceptable level of activity.

[0242] The single chain antigen binding polypeptide can be reacted withthe activated branched polyethylene glycol polymers discussed above inan aqueous reaction medium which can be buffered, depending on the pHrequirements of the nucleophile. The optimum pH for the reaction isgenerally between about 6.5 and about 8.0 and preferably about 7.4 forpolypeptides. The optimum reaction conditions for the sFv stability,reaction efficiency, etc., is within the level of ordinary skill in theart. The preferred temperature range is between 4° C. and 37° C. Thereaction temperature cannot exceed the temperature at which thenucleophile may denature or decompose. It is preferred that thenucleophile be reacted with an excess of the activated branched polymer.Following the reaction, the cojugate is recovered and purified, forexample, by diafiltration, column chromatography, combinations thereof,or the like.

[0243] Conjugates

[0244] Upon production of the polyalkylene oxide conjugated sFv (SCA) ofthe present invention, the polyalkylene oxide conjugated sFv may furtherbe modified by conjugating a diagnostic or therapeutic agent to thepolyalkylene oxide conjugated sFv. The general method of preparing anantibody conjugate according to the invention is described in Shih, L.B., et al., Cancer Res. 51:4192 (1991); Shih, L. B., and D. M.Goldenberg, Cancer Immunol. Immunother. 31:197 (1990); Shih, L. B., etal., Intl. J Cancer 46:1101 (1990); Shih, L. B., et al., Intl. J. Cancer41:832 (1988), all incorporated herein by reference. The indirect methodinvolves reacting an antibody (or sFv), whose polyalkylene oxide has afunctional group, with a carrier polymer loaded with one or plurality ofbioactive molecules, such as, peptides, lipids, nucleic acids (i.e.,phosphate-lysine complexes), drug, toxin, chelator, boron addend ordetectable label molecule(s).

[0245] Alternatively, the polyalkylene oxide conjugated sFv may bedirectly conjugated with a diagnostic or therapeutic agent. The generalprocedure is analogous to the indirect method of conjugation except thata diagnostic or therapeutic agent is directly attached to an oxidizedsFv component. See Hansen et al., U.S. Pat. No. 5,443,953, incorporatedherein by reference.

[0246] The polyalkylene oxide conjugated sFv can be attached to aderivative of the particular drug, toxin, chelator, boron addend orlabel to be loaded, in an activated form, preferably acarboxyl-activated derivative, prepared by conventional means, e.g.,using dicyclohexylcarbodiimide (DCC) or a water soluble variant thereof,to form an intermediate adduct.

[0247] Many drugs and toxins are known which have a cytotoxic effect ontumor cells or microorganisms that may infect a human and cause alesion, in addition to the specific illustrations given above. They areto be found in compendia of drugs and toxins, such as the Merck Indexand the like. Any such drug can be loaded onto a carrier or directlyonto a polyalkylene oxide conjugated sFv by conventional means wellknown in the art, and illustrated by analogy to those described above.

[0248] Chelators for radiometals or magnetic resonance enhancers arealso well known in the art. Typical are derivatives ofethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaaceticacid (DTPA). These typically have groups on the side chain by which thechelator can be attached to a carrier or directly onto a polyalkyleneoxide conjugated sFv. Such groups include, e.g., a benzylisothiocyanate,by which the DTPA or EDTA can be coupled to the reactive group of ansFv.

[0249] Labels such as radioisotopes, enzymes, fluorescent compounds,electron transfer agents, and the like can also be linked to carrier ordirectly onto a polyalkylene oxide conjugated sFv by conventionalmethods well known to the art. These labels and the sFv conjugatesprepared from them can be used for immunoassays and for immunohistology,much as the sFv conjugate prepared by direct attachment of the labels tothe sFv. However, the loading of the conjugates according to the presentinvention with a plurality of labels can increase the sensitivity ofassays or histological procedures, where only low extent of binding ofthe sFv to target antigen is achieved.

[0250] Boron addends, e.g., carboranes, when attached to single-chainantigen binding molecules and targeted to lesions, can be activated bythermal neutron irradiation and converted to radioactive atoms whichdecay by alpha emission to produce highly cytotoxic short-range effects.High loading of boron addends, as well as of magnetic resonanceenhancing ions, is of great importance in potentiating their effects.Carboranes can be made with carboxyl functions on pendant side chains,as is well known in the art.

[0251] Loading of drugs on the carrier will depend upon the potency ofthe drug, the efficiency of sFv targeting and the efficacy of theconjugate once it reaches its target. In most cases, it is desirable toload at least 20, preferably 50, and often 100 or more molecules of adrug on a carrier. The ability to partially, or completely detoxify adrug as a conjugate according to the invention, while it is incirculation, can reduce systemic side effects of the drug and permit itsuse when systemic administration of the unconjugated drug would beunacceptable. Administration of more molecules of the drug, butconjugated to the sFv on a carrier, according to the present invention,permits therapy while mitigating systemic toxicity.

[0252] Toxins will often be less heavily loaded than drugs, but it willstill be advantageous to load at least 5, preferably 10 and in somecases 20 or more molecules of toxin on a carrier and load at least onecarrier chain on the sFv for targeted delivery.

[0253] Uses

[0254] The polyalkylene oxide conjugated sFv (SCA) polypeptideconjugates of the present invention are expected to have much longercirculating half lifes and reduced immunogenicity in vivo. This maysolve a potential limitation relating to very rapid blood clearance ofsome sFv proteins. It would also reduce or eliminate concerns aboutrepeated administration of a therapeutic sFv which may otherwise provokean immune response in the patient. The choice of the particular cysteineand/or oligo-lysine mutant combinations may allow one to achievecirculating lives over a considerable range depending on the specificpolyalkylene oxide conjugated sFv variant polypeptide. This would allowsFv to be administered for the therapeutic use of choice.

[0255] A diagnostic or therapeutic agent is a molecule or atom which isconjugated to an antibody and useful for diagnosis or for therapy. Theimmunoreactivity of the antibody is retained. Diagnostic or therapeuticagents include drugs, toxins, chelators, boron compounds and detectablelabels. See “Conjugates” section, supra, for further details.

[0256] The diagnostic or therapeutic agent may be, but is not limitedto, at least one selected from a nucleic acid, a compound, a protein, anelement, a lipid, an antibody, a saccharide, an isotope, a carbohydrate,an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectableprobe, or any combination thereof, which may be detectably labeled asfor labeling antibodies, as described herein. Such labels include, butare not limited to, enzymatic labels, radioisotope or radioactivecompounds or elements, fluorescent compounds or metals, chemiluminescentcompounds and bioluminescent compounds. Alternatively, any other knowndiagnostic or therapeutic agent can be used in a method of the presentinvention.

[0257] A therapeutic agent used in the present invention may have atherapeutic effect on the target cell, the effect selected from, but notlimited to, correcting a defective gene or protein, a drug action, atoxic effect, a growth stimulating effect, a growth inhibiting effect, ametabolic effect, a catabolic affect, an anabolic effect, an antiviraleffect, an antibacterial effect, a hormonal effect, a neurohumoraleffect, a cell differentiation stimulatory effect, a celldifferentiation inhibitory effect, a neuromodulatory effect, anantineoplastic effect, an anti-tumor effect, an insulin stimulating orinhibiting effect, a bone marrow stimulating effect, a pluripotent stemcell stimulating effect, an immune system stimulating effect, and anyother known therapeutic effects that may be provided by a therapeuticagent delivered to a cell via a delivery system according to the presentinvention.

[0258] The sFv conjugate of the present invention may be used forprotection, suppression or treatment of infection or disease. By theterm “protection” from infection or disease as used herein is intended“prevention,” “suppression” or “treatment.” “Prevention” involvesadministration of a glycosylated sFv conjugate priorto the induction ofthe disease. “Suppression” involves administration of the compositionprior to the clinical appearance of the disease.

[0259] “Treatment” involves administration of the protective compositionafter the appearance of the disease. It will be understood that in humanand veterinary medicine, it is not always possible to distinguishbetween “preventing” and “suppressing” since the ultimate inductiveevent or events may be unknown, latent, or the patient is notascertained until well after the occurrence of the event or events.Therefore, it is common to use the term “prophylaxis” as distinct from“treatment” to encompass both “preventing” and “suppressing” as definedherein. The term “protection,” as used herein, is meant to include“prophylaxis.”

[0260] Such additional therapeutic agents which can further comprise atherapeutic agent or composition of the present invention may beselected from, but are not limited to, known and new compounds andcompositions including antibiotics, steroids, cytotoxic agents,vasoactive drugs, antibodies and other therapeutic modalities.Non-limiting examples of such agents include antibiotics used in thetreatment of bacterial shock, such as gentamycin, tobramycin, nafcillin,parenteral cephalosporins, etc; adrenal corticosteroids and analogsthereof, such as methyl prednisolone, mitigate the cellular injurycaused by endotoxins; vasoactive drugs, such as alpha receptor blockingagent (e.g., phenoxybenzamine), beta receptor agonists (e.g.,isoproterenol), and dopamine are agents suitable for treating septicshock.

[0261] Polyalkylene oxide conjugated sFv of the invention may also beused for diagnosis of disease and to monitor therapeutic response. Otheruses of polyalkylene oxide conjugated sFv proteins are specifictargeting of pro-drug activating enzymes to tumor cells by a bispecificmolecule with specificity for tumor cells and enzyme. Polyalkylene oxideconjugated sFv may be used for specific delivery of drug to an in vivotarget, such as a tumor, delivery of radioactive metals for tumorradioimmunodiagnosis or radioimmunotherapy (Goldenberg, D. M., Am. JMed. 94:297 (1993)), nonradioactive metals in applications such as withboron/uranium-neutron capture therapy (Ranadive, G. N., et al., Nucl.Med. Biol. 20:1 (1993); Barth, R. F., et al., Bioconjug. Chem. 5:58(1994)), and nuclear magnetic resonance imaging (Sieving, P. F., et al.,Bioconjug. Chem. 1:65 (1990)). This list is illustrative only.

[0262] The invention also extends to uses for the polyalkylene oxideconjugated sFv proteins in purification and biosensors. Affinitypurification is made possible by affixing the polyalkylene oxideconjugated sFv protein to a support, with the antigen-binding sitesexposed to and in contact with the ligand molecule to be separated, andthus purified. Biosensors generate a detectable signal upon binding of aspecific antigen to an antigen-binding molecule, with subsequentprocessing of the signal. Polyalkylene oxide conjugated sFv proteins,when used as the antigen-binding molecule in biosensors, may changeconformation upon binding, thus generating a signal that may bedetected.

[0263] The invention is also directed to a method of detecting anantigen suspected of being in a sample by contacting the sample with thepolyalkylene oxide conjugated sFv that is labeled. A sample may compriseat least one compound, mixture, surface, solution, emulsion, suspension,mixture, cell culture, fermentation culture, cell, tissue, secretionand/or derivative or extract thereof.

[0264] Such samples can also include, e.g., animal tissues, such asblood, lymph, cerebrospinal fluid (CNS), bone marrow, gastrointestinalcontents, and portions, cells or internal and external secretions ofskin, heart, lung and respiratory system, liver, spleen, kidney,pancreas, gall bladder, gastrointestinal tract, smooth, skeletal orcardiac muscle, circulatory system, reproductive organs, auditorysystem, the autonomic and central nervous system, and extracts or cellcultures thereof. Such samples can be measured using methods of thepresent invention in vitro, in vivo and in situ.

[0265] Such samples can also include environmental samples such asearth, air or water samples, as well as industrial or commercial samplessuch as compounds, mixtures, surfaces, aqueous chemical solutions,emulsions, suspensions or mixtures.

[0266] Additionally, samples that can be used in methods of the presentinvention include cell culture and fermentation media used for growth ofprokaryotic or eukaryotic cells and/or tissues, such as bacteria, yeast,mammalian cells, plant cells and insect cells.

[0267] Essentially all of the uses for which monoclonal or polyclonalantibodies, or fragments thereof, have been envisioned by the prior art,can be addressed by the polyalkylene oxide conjugated sFv proteins ofthe present invention. These uses include detectably-labeled forms ofthe polyalkylene oxide conjugated sFv protein. Types of labels arewell-knownto those of ordinary skill inthe art. They includeradiolabeling, chemiluminescent labeling, fluorochromic labeling, andchromophoric labeling. Other uses include imaging the internal structureof an animal (including a human) by administering an effective amount ofa labeled form of the polyalkylene oxide conjugated sFv protein andmeasuring detectable radiation associated with the animal. They alsoinclude improved immunoassays, including sandwich immunoassay,competitive immunoassay, and other immunoassays wherein the labeledantibody can be replaced by the PEGylated sFv protein of this invention.See, e.g., Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J.Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, pp.563-681, Elsevier, N (1981); Sambrook et al., Molecular Cloning—ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1989).

[0268] Administration

[0269] Administration of polyalkylene oxide conjugated sFv conjugates ofthe invention for in vivo diagnostic and therapeutic applications willbe by analogous methods to sFv where the diagnostic or therapeuticprinciple is directly linked to the sFv or a loaded carrier is linked byrandom binding to amine or carboxyl groups on amino acid residues of thesFv in a non-site-specific manner.

[0270] Conjugates of the present invention (immunoconjugates) can beformulated according to known methods to prepare pharmaceutically usefulcompositions, such as by admixture with a pharmaceutically acceptablecarrier vehicle. Suitable vehicles and their formulation are described,for example, in Remington's Pharmaceutical Sciences, 18th ed., Osol, A.,ed., Mack, Easton Pa. (1990). In order to form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain atherapeutically effective amount of theimmunoconjugate, either alone, or with a suitable amount of carriervehicle.

[0271] Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the immunoconjugate of thepresent invention. The controlled delivery may be exercised by selectingappropriate macromolecules (for example, polyesters, polyamino acids,polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate). The rate of drug releasemay also be controlled by altering the concentration of suchmacromolecules. Another possible method for controlling the duration ofaction comprises incorporating the therapeutic agents into particles ofa polymeric substance such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, itis possible to entrap the immunoconjugate of the invention inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, by the useofhydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Suchteachings are disclosed in Remington's Pharmaceutical Sciences, 16thed., Osol, A., ed., Mack, Easton Pa. (1990).

[0272] The immunoconjugate may be provided to a patient by means wellknown in the art. Such means of introduction include oral means,intranasal means, subcutaneous means, intramuscular means, intravenousmeans, intra-arterial means, or parenteral means. Intravenous,intraarterial or intrapleural administration is normally used for lung,breast, and leukemic tumors. Intraperitoneal administration is advisedfor ovarian tumors. Intrathecal administration is advised for braintumors and leukemia. Subcutaneous administration is advised forHodgkin's disease, lymphoma and breast carcinoma. Catheter perfusion isuseful for metastatic lung, breast or germ cell carcinomas of the liver.Intralesional administration is useful for lung and breast lesions.

[0273] For therapeutic or diagnostic applications, compositionsaccording to the invention may be administered parenterally incombination with conventional injectable liquid carriers such as sterilepyrogen-free water, sterile peroxide-free ethyl oleate, dehydratedalcohol, or propylene glycol. Conventional pharmaceutical adjuvants forinjection solution such as stabilizing agent, solubilizing agents andbuffers, such as ethanol, complex forming agents such as ethylenediamine tetraacetic acid, tartrate and citrate buffers, andhigh-molecular weight polymers such as polyethylene oxide for viscosityregulation may be added. Such compositions may be injectedintramuscularly, intraperitoneally, or intravenously.

[0274] Further non-limiting examples of carriers and diluents includealbumin and/or other plasma protein components such as low densitylipoproteins, high density lipoproteins and the lipids with which theseserum proteins are associated. These lipids include phosphatidylcholine, phosphatidyl serine, phosphatidyl ethanolamine and neutrallipids such as triglycerides. Lipid carriers also include, withoutlimitation, tocopherol.

[0275] At least one polyalkylene oxide conjugated sFv linked to atherapeutic agent according to the invention may be administered by anymeans that achieve their intended purpose, for example, to treat variouspathologies, such as cell inflammatory, allergy, tissue damage or otherrelated pathologies.

[0276] A typical regimen for preventing, suppressing, or treatingvarious pathologies comprises administration of an effective amount ofan sFv conjugate, administered over a period of one or several days, upto and including between one week and about 24 months.

[0277] It is understood that the dosage of the present inventionadministered in vivo or in vitro will be dependent upon the age, sex,health, and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired. Theranges of effective doses provided below are not intended to limit theinvention and represent preferred dose ranges. However, the mostpreferred dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art, without undueexperimentation. See, e.g., Berkow et al., eds., Merck Manual, 16thedition, Merck and Co., Rahway, N.J. (1992); Goodman et al., eds.,Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8thedition, Pergamon Press, Inc., Elmsford, N.Y. (1990); Avery's DrugTreatment: Principles and Practice ofClinical Pharmacology andTherapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins,Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co.,Boston (1985), Katzung, Basic and Clinical Phamacology, Appleton andLange, Norwalk, Conn. (1992), which references and references citedtherein, are entirely incorporated herein by reference.

[0278] The total dose required for each treatment may be administered bymultiple doses or in a single dose. Effective amounts of adiagnostic/pharmaceutical compound or composition of the presentinvention are from about 0.001 μg to about 100 mg/kg body weight,administered at intervals of 4-72 hours, for a period of 2 hours to 5years, or any range or value therein, such as 0.01-1.0, 1.0-10, 10-50and 50-100 mg/kg, at intervals of 1-4, 6-12, 12-24 and 24-72 hours, fora period of 0.5, 1.0-2.0, 2.0-4.0 and 4.0-7.0 days, or 1, 1-2, 2-4, 4-52or more weeks, or 1, 2, 3-10, 10-20, 20-60 or more years, or any rangeor value therein.

[0279] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions, which maycontain auxiliary agents or excipients which are known in the art.Pharmaceutical compositions such as tablets and capsules can also beprepared according to routine methods. See, e.g., Berker, supra,Goodman, supra, Avery, supra and Ebadi, supra, which are entirelyincorporated herein by reference, including all references citedtherein.

[0280] Pharmaceutical compositions comprising at least one type of sFvconjugate of the invention, or, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 types ofsFv conjugates, of the present invention may be contained in an amounteffective to achieve its intended purpose. In addition to at least onesFv conjugate, a pharmaceutical composition may contain suitablepharmaceutically acceptable carriers, such as excipients, carriersand/or auxiliaries which facilitate processing of the active compoundsinto preparations which can be used pharmaceutically.

[0281] Pharmaceutical compositions may also include suitable solutionsfor administration intravenously, subcutaneously, dermally, orally,mucosally or rectally, and contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active component (i.e., thesFv) together with the excipient. Pharmaceutical compositions for oraladministration include tablets and capsules. Compositions which can beadministered rectally include suppositories. See, e.g., Berker, supra,Goodman, supra, Avery, supra and Ebadi, supra. Additional lipid andlipoprotein drug delivery systems that may be included herein aredescribed more fully in Annals N. Y Acad. Sci. 507:775-88, 98-103, and252-271, which disclosure is hereby incorporated by reference.

[0282] The compositions may also be formulated into orally administrablecompositions containing one or more physiologically compatible carriersor excipients, and may be solid or liquid in form. These compositionsmay, ifdesired, contain conventional ingredients such as binding agents,for example, syrups, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; fillers, such as lactose, mannitol, starch,calcium phosphate, sorbitol, cyclodextran, or methylcellulose;lubricants such as magnesium stearate, high molecular weight polymerssuch as polyethylene glycols, high molecular weight fatty acids such asstearic acid or silica; disintegrants such as starch; acceptable wettingagents as, for example, sodium lauryl sulfate.

[0283] The oral compositions may assume any convenient form, such astablets, capsules, lozenges, aqueous or oily suspensions, emulsions, ordry products suitable for reconstitution with water or other liquidmedium prior to use. The liquid oral forms may, of course, containflavors, sweeteners, preservatives such as methyl or propylp-hydroxybenzoates; suspending agents such as sorbitol, glucose or othersugar syrup, methyl, hydroxymethyl, or carboxymethyl celluloses orgelatin; emulsifying agents such as lecithin or sorbitan monooleate orthickening agents. Non-aqueous compositions may also be formulated whichcomprise edible oils as, for example, fish-liver or vegetable oils.These liquid compositions may conveniently be encapsulated in, forexample, gelatin capsules in a unit dosage amount.

[0284] The pharmaceutical compositions according to the presentinvention may also be administered, if appropriate, either topically asan aerosol or, formulated with conventional bases as a cream orointment.

[0285] The pharmaceutical compositions of the present invention can alsobe administered by incorporating the active ingredient into colloidalcarriers, such as liposomes. Liposome technology is well known in theart, having been described by Allison et al., Nature 252:252-254 (1974),and Dancy et al., J. Immunol. 120:1109-1113 (1978).

[0286] Having now generally described this invention, the same will bebetter understood by reference to certain specific examples, which areincluded for the purpose of illustration and not intended to be limitingunless otherwise specified.

EXAMPLES Example 1 Preparation of Methoxypoly(ethyleneglycol)-succinimidyl Carbonate (SC-PEG)

[0287] Dissolve 60 g of methoxy poly(ethylene glycol) (MW 5,000) in 200rnl of 3/1 toluene/dichloromethane and treat with atoluene solution ofphosgene (30 ml, 57 mmol) overnight. Evaporate the solutionto drynessand remove the remainder of the phosgene under vacuum. Redissolve theresidue in 150 ml of 2/1 toluene/dichloromethane. Treat the resultingsolution with 2.1 g (18 mmol) of solid N-hydroxysuccinimide, followed by1.7 ml (12 mmol) of triethylamine. Allow the solution to stand for threehours and then filter it and evaporate it to dryness. Dissolve theresidue in 600 ml of warm (50° C.) ethyl acetate, filter the solution,and cool it to facilitate precipitation of the polymer. Collect theproduct by filtration, then recrystallize from ethyl acetate, and dryunder vacuum over P₂O₅.

Example 2 Preparation of CC 49/212 SCA

[0288] In the production of monovalent or multivalent antigen-bindingproteins, the same recombinant E. coli production system that was usedfor prior single-chain antigen-binding protein production was used. SeeBird et al., Science 242:423 (1988). This production system producedbetween 2 and 20% of the total E. coli protein as single-chainantigen-binding protein. For protein recovery, the frozen cell pastefrom three 10-liter fermentations (600-900 g) was thawed overnight at 4°C. and gently resuspended at 4° C. in 50 mM Tris-HCl, 1.0 mM EDTA, 100mM KCl, 0.1 mM PMSF, pH 8.0 (lysis buffer), using 10 liters of lysisbuffer for every kilogram of wet cell paste. When thoroughlyresuspended, the chilled mixture was passed three times through aManton-Gaulin cell homogenizer to totally lyse the cells. Because thecell homogenizer raised the temperature of the cell lysate to 25±5° C.,the cell lysate was cooled to 5±2° C. with a Lauda/Brinkman chillingcoil after each pass. Complete lysis was verified by visual inspectionunder a microscope.

[0289] The cell lysate was centrifuged at 24,300 g for 30 minutes at 6°C. using a Sorvall RC-5B centrifuge. The pellet containing the insolublesingle-chain antigen-binding protein was retained, and the supernatantwas discarded. The pellet was washed by gently scraping it from thecentrifuge bottles and resuspending it in 5 liters of lysis buffer/kg ofwet cell paste. The resulting 3.0-to 4.5-liter suspension was againcentrifuged at 24,300 g for 30 minutes at 6° C., and the supernatant wasdiscarded. This washing of the pellet removes soluble E. coli proteinsand can be repeated as many as five times. At any time during thiswashing procedure the material can be stored as a frozen pellet at −20°C. A substantial time saving in the washing steps can be accomplished byutilizing a Pellicon tangential flow apparatus equipped with 0.22-μmmicroporous filters, in place of centrifugation.

[0290] The washed pellet was solubilized at 4° C. in freshly prepared 6M guanidine hydrochloride, 50 mM Tris-HCl, 10 mM CaCl₂, 50 mM KCl, pH8.0 (dissociating buffer), using 9 ml/g of pellet. If necessary, a fewquick pulses from a Heat Systems Ultrasonics tissue homogenizer can beused to complete the solubilization. The resulting suspension wascentrifuged at 24,300 g for 45 minutes at 6° C. and the pellet wasdiscarded. The optical density of the supernatant was determined at 280nm and if the OD₂₈₀ was above 30, additional dissociating buffer wasadded to obtain an OD₂₈₀ of approximately 25.

[0291] The supernatant was slowly diluted into cold (4-7° C.) refoldingbuffer (50 mM Tris-HCl, 10 mM CaCl₂, 50 mM KCl, pH 8.0) until a 1:10dilution was reached (final volume 10-20 liters). Re-folding occurs overapproximately eighteen hours under these conditions. The best resultsare obtained when the GuHCl extract is slowly added to the refoldingbuffer over a two hour period, with gentle mixing. The solution wasfiltered through a 0.2 μm Millipore Millipak 200. This filtration stepmay be optionally preceded by a centrifugation step. The filtrate wasconcentrated to 1 to 2 liters using an Amicon spiral cartridge with10,000 MWCO cartridge, again at 4° C.

[0292] The concentrated crude antigen-binding protein sample wasdialyzed against Buffer G (60 mM MOPS, 0.5 mM Ca acetate, pH 6.0-6.4)until the conductivity was lowered to that of Buffer G. The sample wasthen loaded on a 21.5×250-mm polyaspartic acid PolyCAT A column,manufactured by Poly LC of Columbia, Md. If more than 60 mg of proteinis loaded on this column, the resolution begins to deteriorate; thus,the concentrated crude sample often must be divided into several PolyCATA runs. Most antigen-binding proteins have an extinction coefficient ofabout 2.0 ml mg⁻¹cm⁻¹ at 280 nm and this can be used to determineprotein concentration. The antigen-binding protein sample was elutedfrom the PolyCAT A column with a 50-min linear gradient from Buffer G toBuffer H (60 mM MOPS, 20 mM Ca Acetate, pH 7.5-8.0). Most of thesingle-chain proteins elute between 20 and 26 minutes when this gradientis used. This corresponds to an eluting solvent composition ofapproximately 70% Buffer G and 30% Buffer H. Most of the bivalentantigen-binding proteins elute later than 45 minutes, which correspondto over 90% Buffer H.

Example 3 Modification of CC 49/212 Single Chain Antigen BindingMolecule With SC-PEG

[0293] A sample of CC 49/212 single chain antigen binding molecule(MW=27000) dissolved in KPO₄/NaCl buffer (pH 7.2) was obtained asdescribed in Example 2. The protein was found to be pure using SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and sizeexclusion chromatography. The concentration of the protein was 0.79mg/ml. It was further concentrated to at least 2 mg/ml using an Amiconconcentrator with a 10,000 dalton nominal size cut-off, i.e., anythinggreater than 10K is retained.

[0294] The modification reaction, i.e., the coupling of the SC-PEG tothe CC 49/212, was carried out in 50 mM KPO₄, 150 mM NaCl buffer, whichwas the storage buffer the protein was supplied in. The pH was raisedfrom 7.2 to 7.5. SC-PEG (MW 5,000) was added in a 50× molar excess toprotein. At specific time intervals, the coupling reaction wasterminated by the addition of a 50× molar excess of glycine and theextent and progress of the coupling reaction was checked as a functionof time using both size exclusion chromatography using a DuPont Zorbax250 column and SDS-PAGE.

[0295] Free SC-PEG remaining in the samples was removed by extensivedialysis on an Amicon Centricon 10.

[0296] The samples were checked for degree of modification by sizeexclusion chromatography. After concentrating the samples, the residualamine concentration on the protein was determined by titration withtrinitrobenzene sulfonate and the percentage of amine groups that hadreacted with the SC-PEG (the “% modification”) was calculated from theresults.

[0297] Dansyl derivatives of native single chain antigen bindingmolecule (CC 49/212) and hemoglobin, PEG SCA and hemoglobin, andN-acetyl lysine were prepared. These samples were then analyzed foramino acid. The results of this experiment are shown in Table 3. TABLE 3Reaction Time Protein Molecular Weight¹ (Minutes) (mg/ml) % Modification(Kilodaltons)  0 0.74148  0  27  15 1.3569 52  84  30 1.3587 62 156  601.2706 63  80 224  90 247 120 0.78 65

Example 4 Competition ELISA

[0298] The CC49 monoclonal antibody was developed by Dr. JeffreySchlom's group, Laboratory of Tumor Immunology and Biology, NationalCancer Institute. It binds specifically to the pan-carcinoma tumorantigen TAG-72. See Muraro, R. et al., Cancer Research 48: 4588-4596(1988).

[0299]FIG. 1 is a graphical representation of three competition ELISA'sin which unlabeled PEG modified CC49/212 single-chain Fv (closedsquares), CC49/212 single-chain Fv (open squares), CC49 IgG (opencircles), and MOPC-21 IgG (+) competed against a CC49 IgG radiolabeledwith ¹²⁵I for binding to the TAG-72 antigen on a human breast melanomaextract. MOPC-21 is a control antibody that does not bind to TAG-72antigen. In this experiment, 50% competition of ¹²⁵I-CC49 IgG bindingrequired about 200 nM of CC49 IgG, about 550 nM of CC49/212 sFv, andabout 3000 nM of PEG modified CC49/212 sFv.

Example 5 Preparation of U-PEG-OH

[0300]

[0301] Materials

[0302] Methoxypoly(ethylene glycol) (m-PEG) was obtained from UnionCarbide. The solvents were obtained from Aldrich Chemical of Milwaukee,Wis. The methoxypoly(ethylene glycol)-N-succinimidyl carbonate (SC-PEG)was prepared as described in U.S. Pat. No. 5,122,614, using m-PEG havinga molecular weight of about 5,000. Each of the products prepared inExamples 5-10 was confirmed structurally by carbon-13 NMR.

[0303] The branched polymer, U-PEG-OH, was prepared by adding 100 mg(1.1 mmol) of 1,3-diamino-2-propanol to a solution of 10.0 g (2 mmol) ofSC-PEG in 50 mL of methylene chloride. The mixture was stirred for 18hours at room temperature then filtered. Excess solvent was removed bydistillation in vacuo. The residue was recrystallized from 2-propanol toyield 7.1 g of product (70% yield).

Example 6 Preparation of U-PNP-PEG

[0304]

[0305] The compound of Example 5 was activated with p-nitrophenylchloroformate. First, 5.0 g (0.5 mmol) of U-PEG-OH was azeotropicallydried by refluxing in 75 mL of toluene for 2 hours, resulting in theremoval of 25 mL of solvent/water. The reaction mixture was cooled to30° C., followed by the addition of 120 mg (0.6 mmol) of p-nitrophenylchloroformate and 50 mg (0.6 mmol) of pyridine. The resulting mixturewas stirred for two hours at 45° C., followed by stirring overnight atroom temperature.

[0306] The reaction mixture was then filtered through CELITE™, followedby removal of the solvent from the filtrate by distillation in vacuo.The residue was recrystallized from 2-propanol to yield 4.2 g (81%yield) of the product.

Example 7 Preparation of US-PEG

[0307]

[0308] In this example, the U-PNP-PEG of Example 6 was reacted withN-hydroxysuccinimide to form the succinimidyl carbonate ester of U-PEG.A solution containing 5.0 g (0.5 mmol) of the U-PNP-PEG, 0.6 g (5 mmol)of N-hydroxysuccinimide and 0.13 g (1 mmol) of diisopropylethylamine in40 ml of methylene chloride was refluxed for 18 hours. The solvent wasthen removed by distillation in vacuo, and the residue wasrecrystallized from 2-propanol to yield 4.2 g of the succinimidylcarbonate ester (82% yield).

Example 8 Preparation of XU-PEG-OH

[0309]

[0310] This branched polymer was prepared by reacting the U-PNP-PEG ofExample 6 with 2-(2-aminoethoxy) ethanol (i.e., the amino alcohol wasreacted with the p-nitrophenyl carbonate). The recrystallized productyield was 86%.

Example 9 Preparation of XU-PNP-PEG

[0311] The compound of Example 8 was functionalized with p-nitrophenylcarbonate as in Example 6. The recrystallized product yield was 83%.

Example 10 Preparation of XUS-PEG

[0312]

[0313] In this example, the succinimidyl carbonate derivative ofcompound prepared in Example 8 was prepared according to the processdescribed in Example 7, by reacting N-hydroxysuccinimide with thep-nitrophenyl carbonate derivative of Example 9. The recovered productyield was 84%.

Example 11 Modification of CC49/218 with SC-PEG or XUS-PEG

[0314] A sample containing CC49/218 was desalted on a PD-10 column in abuffer consisting of 0.1M sodium phosphate, pH 8.0. An equimolar amountof SC-PEG or XUS-PEG was added and the reactions were incubated at 4°C., overnight. The reactions were quenched with an excess of glycine.The modified CC49/218 conjugates were GPC purified and then concentratedin a centricon-10.

[0315] The yield based on GPC integration was about 50% for SC-PEGmodified CC49/218 and about 40% for XUS-PEG modified CC49/218. The GPCprofiles were almost identical to those obtained when the reaction wasperformed at pH 9.0, room temperature. SDS-PAGE revealed that theappropriate derivatives had been made.

Example 12 Competition ELISA

[0316] The assay was performed as in Example 4 above using SC-PEGmodified CC49/218 and XUS-PEG modified CC49/218 along with theappropriate controls. The results are shown in FIG. 4 and in Table 4below. Sample 50% Inhibition (nM) CC49 IgG  10 GC49/218 sFv  80 SCUnreacted 300 #049301 SC Reacted 650 #049304 XUS Unreacted 280 #049302XUS Reacted 320 #049303

[0317] Thus, the affinity of the SC-PEG modified CC49-SCA was withinabout 8 to 10 fold of the native CC49-SCA and the affinity the XUS-PEGmodified CC49-SCA was within about 4 to 5 fold of the native CC49-SCA.

[0318] Samples #049304 and #049303 were PEG modified, whiles samples#04901 and #049302 were unmodified CC49/218 isolated from the reactionmixtures.

Example 13 Pharmacokinetics of Plasma Retention of sFv and PEG-sFv

[0319] Sixty μg of CC49/218 sFv protein or 60 μg of PEG-modified sFvprotein were injected intravenously at time 0 into ICR (CD-1) femalemice (Harlan—25 g, 7-8 weeks old). Mice were bled at the time pointsindicated in FIG. 5. The percent retention in plasma was quantitated byELISA methods. For the PEG-modified conjugate, CC49/218 sFv wasconjugated to SC-PEG of molecular mass 20,000 (the protocol is describedin U.S. Pat. No. 5,122,614, which disclosure is incorporated herein byreference). The average PEG:sFv molar ratio in the tested PEG-sFvconjugate was approximately 1:1.

Example 14 PEGylated Multimer Single Chain Antibodies

[0320] Using bifunctional PEG, dimers and trimers of CC49-SCA have beenmade. CC49-SCA was modified with bifunctional PEG as follows: 2 ml ofCC49-SCA in phosphate buffered saline (25 mM sodium phosphate, pH 7.3,0.15 M NaCl) at a concentration of 1.5 mg/ml was modified as follows.Bifunctional PEG (polyethylene glycol with reactive SC at both terminalends), 1.887 mg (powder) was dissolved in 0.1 ml of MOPS(3-[N-morpholino]propane)-sulfonic acid) buffered at pH 7.3. This PEGsolution was added to CC49-SCA solution within 10 seconds ofdissolution. The mixture was then stirred at 24° C. for 1 hour. At theend of the reaction, solid Guanidine HCl was added to the reactionmixture to a final concentration of 6 M in order to break up noncovalently associated CC49-SCA. This material was immediately applied toa size exclusion column (2 cm×60 cm, Superdex-75) previouslyequilibrated in the buffer composed of 60 mM Guanidine HCl in 50 mM TrispH 7.3, 1 mM CaCl₂, 0.1 mM PMSF (phenyl methyl sulfonyl flouride), and50 mM KCl. Multimers of different molecular weights were thenfractionated from the column.

[0321] These multimers are CC49-SCA separated by a long stretch of PEG(5000 MW, about 226 carbons in length) and were freshly refolded andthus, are not believed to arise from aggregation. It is less likely thatthere was diabody or multivalent SCA formed as a result of selfassociation of the CC49-SCA. Further evidence was demonstrated by thefact that there was negligible native CC49-SCA in the denaturingSDS-PAGE profile.

[0322] The SDS-PAGE electrophoresis patterns for dimeric CC49-SCA,trimeric CC49-SCA, PEG-CC49-SCA and native CC49-SCA under reducingconditions are shown in FIG. 6.

[0323] The multimers were assayed for binding affinity using thefollowing assay which was modified method described in B Friquet et al.J. of Immunology Methods, 77:305-319(1985). Briefly, various amounts ofa given modified single chain antibody were mixed with various amountsof the antigen mucin in PBS (phosphate buffered saline). The bindingreaction was allowed to reach equilibration for at least 24 hours at 4°C. At the end of the incubation, the unbound CC49-SCA fractions wereassayed by ELISA, while the bound fractions were washed away. The totalamount of free CC49-SCA was determined by the ELISA using the CC49-SCAsample pre-incubated in the absence of the antigen mucin. The boundantibody was determined by subtraction of the free (unbound) amount fromthe total amount as determined by ELISA. Since the total amount of theCC49-SCA was known, it was also used as its own standard curve. Notethat each type of CC49-SCA had its own reference control. In essence,the protocol was measuring the unbound amount of a particular version ofPEG-CC49-SCA as a result of the binding to the antigen. Although PEG mayaffect the detection reagent in ELISA, this was well contained in thestandard references. Therefore, the amount measured was not due to thedifference of various PEG on the various versions of PEG-CC49-SCA.

[0324] In sum, in this study, increasing concentrations of the CC49-SCAprotein were allowed to bind to a fixed amount of the antigen. Theamount that binds to 50% of the maximal level is a good indication ofthe affinity. This data indicate that the affinity of PEG-Di-CC49-SCAand PEG-Tri-CC49-SCA are very similar to that of native CC49-SCA.However, the PEG-modified CC49-SCA monomer had much lower affinity. Thebinding data are shown in FIG. 7.

Example 15 Pharmacokinetics of PEG-CC49-SCA

[0325] The pharmacokinetic study of various forms of PEG-CC49-SCA wasperformed as in Example 13, above.

[0326] The data obtained from this study indicate the following:

[0327] There was a trend toward longer circulation half-lives as thesize of the PEG increases. As more PEG is attached to the protein, thecirculation time increases. However, attachment of a few strands of highmolecular weight PEG gives a better increase in circulation half-lifethan multiple stands of lower molecular weight PEG.

[0328] The circulation half-life of CC49-SCA-U-PEG, made with the US-PEGprepared in Example 7, was about the same as that of CC49-SCA-PEG-12000.Therefore, the shape of the PEG does not affect the circulationhalf-life.

[0329] Whether the linker is an SC-bond, Flan-bond, hydrazine bond, orTPC, there was no significant change inthe circulation half-life.Therefore, the chemical bonds of the linkers, if not releasable, do notaffect the circulation half-life. In addition, the PEG remains attachedto the protein during the observable time.

[0330] The circulation half-life was shortened by carbohydrate. However,if PEG was attached to the carbohydrate, it increases the circulation byabout 10 fold. This was not better, however, than attaching anequivalent number of PEG at other sites on CC49-SCA.

[0331] The results of the study are shown in the table below:Pharmacokinetic Data of SCA and PEG-SCA Native CC49 Circulatory AreaUnder the Mean Residence or PEG used # PEG half-life (hours) Curve(μg/ml-hour) Time (hours) MW Linker Label per SCA Mean Std. Error MeanStd. Error Mean Std. Error Native CC49/218 0 0.69 0.41 54 27 1 0.592,000 SC 2.1 3.41 0.38 135.91 13.02 4.92 0.55 3,400 NHS biotin 2 2.520.45 95.03 14.48 3.64 0.65 5,000 Flan 1 1.81 0.44 67.41 13.98 2.61 0.635,000 SC 1.7 4.15 1.08 147.96 32.7 5.98 1.56 5,000 TPC 1.4 3.48 0.95120.56 28.05 5.02 1.37 5,000 Hz 4.8 9.81 1.61 395.33 56.32 14.15 2.3210,000 U biotin 1.5 10.42 1.36 415.86 47.21 15.03 1.96 12,000 Flan 1.213.98 1.76 583.19 64.47 20.17 2.53 12,000 SC 1.3 13.27 1.5 586.05 58.0819.15 2.16 20,000 SC 1 12.86 0.73 1,111 56 18.55 1.05 Native gCC49/3 00.39 0.01 36 1 0.56 0.02 5,000 Hz 4.2 3.84 0.46 305 31 5.54 0.66

Example 16

[0332] Competition Binding Assay

[0333] A competition binding assay of biotinylated CC49-SCA with variousPEGylated CC49-SCA proteins was performed using an ELISA of thebiotinylated CC49-SCA as detected by horseradish-peroxidase conjugatedwith streptoavidin (SAV-HRP). In the assay, the biotinylated CC49-SCAand PEG-CC49-SCA sample were mixed at various ratios for competition ofbinding to the antigen mucin on a surface. The amount ofbiotinylated-CC49-SCA bound to antigen was then measured by SAV-HRP,which would not detect the PEG-modified CC49-SCA. The reduction ofbinding of biotinylated CC49-SCA due to competition of PEG-CC49-SCA is areflection of the relative affinity of the two forms of CC49-SCA for theantigen. The level of PEG-CC49-SCA that caused reduction ofbiotin-CC49-SCA to half of its maximum binding level (IC50) was used inthe Cheng-Prussoff formula for determination of the affinity constant Kdas follows: Assuming the biotin-CC49-SCA level is [s] and its affinityis known as Ks, then the affinity for the PEG-CC49-SCA is Kd=IC50/(1+[s]/Ks). Note that PEG-CC49-SCA was not directly measured andthat PEG had no effect on biotin binding because it was on a differentmolecule. The estimated Kd was then expressed as a percentage of thecontrol (CC49-SCA).

[0334] The affinity ranking obtained was as follows:

[0335] CC49=SC2>GC=Bio.CC49=CC12>C20=F5>HZ>PG

[0336] where SC2 is PEG SC2000-CC49-SCA; GC is glyco-CC49-S CA;Bio.CC49-SCA is the biotinylated CC49-SCA; CC12 is thePEG-SC12,000-CC49-SCA; F5 is PEG-Flan-5000-CC49-SCA; HZ is CC49-SCAhighly PEGylated on the carboxyl groups with MW 5000 hydrazine-PEG; PGis PEG-glyco-CC49-SCA and is highly PEGylated on the carbohydrate withMW 5000 hydrazine PEG; and C20 is PEG-SC20000-CC49-SCA. The affinity forall the PEG-modified CC49-SCAs shown in FIG. 8, were within about twofold of the native CC49-SCA. Taken together with the data in Example 15(see, the table), the results indicate that when the SCA is modifiedwith a lower number of PEG molecules, the resulting affinity of the SCAis better than when the SCA is modified with a higher number of PEGmolecules.

[0337] Although the foregoing refers to particular preferredembodiments, it will be understood that the present invention is not solimited. It will occur to those skilled in the art that variousmodifications may be made to the disclosed embodiments and that suchmodifications are intended to be within the scope of the presentinvention.

1 6 749 base pairs nucleic acid double both cDNA CDS 1..738 1 GAC GTCGTG ATG TCA CAG TCT CCA TCC TCC CTA CCT GTG TCA GTT GGC 48 Asp Val ValMet Ser Gln Ser Pro Ser Ser Leu Pro Val Ser Val Gly 1 5 10 15 GAG AAGGTT ACT TTG AGC TGC AAG TCC AGT CAG AGC CTT TTA TAT AGT 96 Glu Lys ValThr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 GGT AAT CAAAAG AAC TAC TTG GCC TGG TAC CAG CAG AAA CCA GGG CAG 144 Gly Asn Gln LysAsn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 TCT CCT AAA CTGCTG ATT TAC TGG GCA TCC GCT AGG GAA TCT GGG GTC 192 Ser Pro Lys Leu LeuIle Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 CCT GAT CGC TTC ACAGGC AGT GGA TCT GGG ACA GAT TTC ACT CTC TCC 240 Pro Asp Arg Phe Thr GlySer Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 75 80 ATC AGC TGT GTG AAGACT GAA GAC CTG GCA GTT TAT TAC TGT CAG CAG 288 Ile Ser Cys Val Lys ThrGlu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 TAT TAT AGC TAT CCC CTCACG TTC GGT GCT GGG ACC AAG CTT GTG CTG 336 Tyr Tyr Ser Tyr Pro Leu ThrPhe Gly Ala Gly Thr Lys Leu Val Leu 100 105 110 AAA GGC TCT TGT TCC GGTAGC GGC AAA CCC GGG AGT GGT GAA GGT AGC 384 Lys Gly Ser Cys Ser Gly SerGly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 ACT AAA GGT CAG GTT CAGCTG CAG CAG TCT GAC GCT GAG TTG GTG AAA 432 Thr Lys Gly Gln Val Gln LeuGln Gln Ser Asp Ala Glu Leu Val Lys 130 135 140 CCT GGG GCT TCA GTG AAGATT TCC TGC AAG GCT TCT GGC TAC ACC TTC 480 Pro Gly Ala Ser Val Lys IleSer Cys Lys Ala Ser Gly Tyr Thr Phe 145 150 155 160 ACT GAC CAT GCA ATTCAC TGG GTG AAA CAG AAC CCT GAA CAG GGC CTG 528 Thr Asp His Ala Ile HisTrp Val Lys Gln Asn Pro Glu Gln Gly Leu 165 170 175 GAA TGG ATT GGA TATTTT TCT CCC GGA AAT GAT GAT TTT AAA TAC AAT 576 Glu Trp Ile Gly Tyr PheSer Pro Gly Asn Asp Asp Phe Lys Tyr Asn 180 185 190 GAG AGG TTC AAG GGCAAG GCC ACA CTG ACT GCA GAC AAA TCC TCC AGC 624 Glu Arg Phe Lys Gly LysAla Thr Leu Thr Ala Asp Lys Ser Ser Ser 195 200 205 ACT GCC TAC GTG CAGCTC AAC TGC CTG ACA TCT GAG GAT TCT GCA GTG 672 Thr Ala Tyr Val Gln LeuAsn Cys Leu Thr Ser Glu Asp Ser Ala Val 210 215 220 TAT TTC TGT ACA AGATCC CTG AAT ATG GCC TAC TGG GGT CAA GGA ACC 720 Tyr Phe Cys Thr Arg SerLeu Asn Met Ala Tyr Trp Gly Gln Gly Thr 225 230 235 240 TCA GTC ACC GTCTCC TGC TAATAGGATC C 749 Ser Val Thr Val Ser Cys 245 246 amino acidsamino acid linear protein 2 Asp Val Val Met Ser Gln Ser Pro Ser Ser LeuPro Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Leu Ser Cys Lys Ser SerGln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp TyrGln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala SerAla Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Ser 65 70 75 80 Ile Ser Cys Val Lys Thr Glu Asp LeuAla Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe GlyAla Gly Thr Lys Leu Val Leu 100 105 110 Lys Gly Ser Cys Ser Gly Ser GlyLys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Gln Val Gln LeuGln Gln Ser Asp Ala Glu Leu Val Lys 130 135 140 Pro Gly Ala Ser Val LysIle Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145 150 155 160 Thr Asp His AlaIle His Trp Val Lys Gln Asn Pro Glu Gln Gly Leu 165 170 175 Glu Trp IleGly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn 180 185 190 Glu ArgPhe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 195 200 205 ThrAla Tyr Val Gln Leu Asn Cys Leu Thr Ser Glu Asp Ser Ala Val 210 215 220Tyr Phe Cys Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Thr 225 230235 240 Ser Val Thr Val Ser Cys 245 782 base pairs nucleic acid bothboth cDNA CDS 1..771 3 GAC GTC GTG ATG TCA CAG TCT CCA TCC TCC CTA CCTGTG TCA GTT GGC 48 Asp Val Val Met Ser Gln Ser Pro Ser Ser Leu Pro ValSer Val Gly 250 255 260 GAG AAG GTT ACT TTG AGC TGC AAG TCC AGT CAG AGCCTT TTA TAT AGT 96 Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser LeuLeu Tyr Ser 265 270 275 GGT AAT CAA AAG AAC TAC TTG GCC TGG TAC CAG CAGAAA CCA GGG CAG 144 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln LysPro Gly Gln 280 285 290 TCT CCT AAA CTG CTG ATT TAC TGG GCA TCC GCT AGGGAA TCT GGG GTC 192 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg GluSer Gly Val 295 300 305 310 CCT GAT CGC TTC ACA GGC AGT GGA TCT GGG ACAGAT TTC ACT CTC TCC 240 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Ser 315 320 325 ATC AGC AGT GTG AAG ACT GAA GAC CTG GCA GTTTAT TAC TGT CAG CAG 288 Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Val TyrTyr Cys Gln Gln 330 335 340 TAT TAT AGC TAT CCC CTC ACG TTC GGT GCT GGGACC AAG CTT GTG CTG 336 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly ThrLys Leu Val Leu 345 350 355 AAA GGC TCT ACT TCC GGT AGC GGC AAA CCC GGGAGT GGT GAA GGT AGC 384 Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly SerGly Glu Gly Ser 360 365 370 ACT AAA GGT CAG GTT CAG CTG CAG CAG TCT GACGCT GAG TTG GTG AAA 432 Thr Lys Gly Gln Val Gln Leu Gln Gln Ser Asp AlaGlu Leu Val Lys 375 380 385 390 CCT GGG GCT TCA GTG AAG ATT TCC TGC AAGGCT TCT GGC TAC ACC TTC 480 Pro Gly Ala Ser Val Lys Ile Ser Cys Lys AlaSer Gly Tyr Thr Phe 395 400 405 ACT GAC CAT GCA ATT CAC TGG GTG AAA CAGAAC CCT GAA CAG GGC CTG 528 Thr Asp His Ala Ile His Trp Val Lys Gln AsnPro Glu Gln Gly Leu 410 415 420 GAA TGG ATT GGA TAT TTT TCT CCC GGA AATGAT GAT TTT AAA TAC AAT 576 Glu Trp Ile Gly Tyr Phe Ser Pro Gly Asn AspAsp Phe Lys Tyr Asn 425 430 435 GAG AGG TTC AAG GGC AAG GCC ACA CTG ACTGCA GAC AAA TCC TCC AGC 624 Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr AlaAsp Lys Ser Ser Ser 440 445 450 ACT GCC TAC GTG CAG CTC AAC AGC CTG ACATCT GAG GAT TCT GCA GTG 672 Thr Ala Tyr Val Gln Leu Asn Ser Leu Thr SerGlu Asp Ser Ala Val 455 460 465 470 TAT TTC TGT ACA AGA TCC CTG AAT ATGGCC TAC TGG GGT CAA GGA ACC 720 Tyr Phe Cys Thr Arg Ser Leu Asn Met AlaTyr Trp Gly Gln Gly Thr 475 480 485 TCG GTC ACC GTC TCC AAA AAG AAG AAAAAA AAG AAA AAG GTC ACC GTC 768 Ser Val Thr Val Ser Lys Lys Lys Lys LysLys Lys Lys Val Thr Val 490 495 500 TCC TAATAGGATC C 782 Ser 257 aminoacids amino acid linear protein 4 Asp Val Val Met Ser Gln Ser Pro SerSer Leu Pro Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Leu Ser Cys LysSer Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu AlaTrp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr TrpAla Ser Ala Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser GlySer Gly Thr Asp Phe Thr Leu Ser 65 70 75 80 Ile Ser Ser Val Lys Thr GluAsp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu ThrPhe Gly Ala Gly Thr Lys Leu Val Leu 100 105 110 Lys Gly Ser Thr Ser GlySer Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Thr Lys Gly Gln ValGln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys 130 135 140 Pro Gly Ala SerVal Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145 150 155 160 Thr AspHis Ala Ile His Trp Val Lys Gln Asn Pro Glu Gln Gly Leu 165 170 175 GluTrp Ile Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn 180 185 190Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 195 200205 Thr Ala Tyr Val Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 210215 220 Tyr Phe Cys Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Thr225 230 235 240 Ser Val Thr Val Ser Lys Lys Lys Lys Lys Lys Lys Lys ValThr Val 245 250 255 Ser 723 base pairs nucleic acid both both CDS 1..7235 GAC GTC GTG ATG TCA CAG TCT CCA TCC TCC CTA CCT GTG TCA GTT GGC 48 AspVal Val Met Ser Gln Ser Pro Ser Ser Leu Pro Val Ser Val Gly 1 5 10 15GAG AAG GTT ACT TTG AGC TGC AAG TCC AGT CAG AGC CTT TTA TAT AGT 96 GluLys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 GGTAAT CAA AAG AAC TAC TTG GCC TGG TAC CAG CAG AAA CCA GGG CAG 144 Gly AsnGln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 TCT CCTAAA CTG CTG ATT TAC TGG GCA TCC GCT AGG GAA TCT GGG GTC 192 Ser Pro LysLeu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 CCT GAT CGCTTC ACA GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC TCC 240 Pro Asp Arg PheThr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 75 80 ATC AGC AGTGTG AAG ACT GAA GAC CTG GCA GTT TAT TAC TGT CAG CAG 288 Ile Ser Ser ValLys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 TAT TAT AGC TATCCC CTC ACG TTC GGT GCT GGG ACC AAG CTT GTG CTG 336 Tyr Tyr Ser Tyr ProLeu Thr Phe Gly Ala Gly Thr Lys Leu Val Leu 100 105 110 AAA GGC TCT ACTTCC GGT AGC GGC AAA TCC TCT GAA GGC AAA GGT CAG 384 Lys Gly Ser Thr SerGly Ser Gly Lys Ser Ser Glu Gly Lys Gly Gln 115 120 125 GTT CAG CTG CAGCAG TCT GAC GCT GAG TTG GTG AAA CCT GGG GCT TCA 432 Val Gln Leu Gln GlnSer Asp Ala Glu Leu Val Lys Pro Gly Ala Ser 130 135 140 GTG AAG ATT TCCTGC AAG GCT TCT GGC TAC ACC TTC ACT GAC CAT GCA 480 Val Lys Ile Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Asp His Ala 145 150 155 160 ATT CAC TGGGTG AAA CAG AAC CCT GAA CAG GGC CTG GAA TGG ATT GGA 528 Ile His Trp ValLys Gln Asn Pro Glu Gln Gly Leu Glu Trp Ile Gly 165 170 175 TAT TTT TCTCCC GGA AAT GAT GAT TTT AAA TAC AAT GAG AGG TTC AAG 576 Tyr Phe Ser ProGly Asn Asp Asp Phe Lys Tyr Asn Glu Arg Phe Lys 180 185 190 GGC AAG GCCACA CTG ACT GCA GAC AAA TCC TCC AGC ACT GCC TAC GTG 624 Gly Lys Ala ThrLeu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Val 195 200 205 CAG CTC AACAGC CTG ACA TCT GAG GAT TCT GCA GTG TAT TTC TGT ACA 672 Gln Leu Asn SerLeu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Thr 210 215 220 AGA TCC CTGAAT ATG GCC TAC TGG GGT CAA GGA ACC TCA GTC ACC GTC 720 Arg Ser Leu AsnMet Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 225 230 235 240 TCC 723Ser 241 amino acids amino acid linear protein 6 Asp Val Val Met Ser GlnSer Pro Ser Ser Leu Pro Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr LeuSer Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Lys AsnTyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu LeuIle Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe ThrGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 75 80 Ile Ser Ser ValLys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser TyrPro Leu Thr Phe Gly Ala Gly Thr Lys Leu Val Leu 100 105 110 Lys Gly SerThr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly Gln 115 120 125 Val GlnLeu Gln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala Ser 130 135 140 ValLys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His Ala 145 150 155160 Ile His Trp Val Lys Gln Asn Pro Glu Gln Gly Leu Glu Trp Ile Gly 165170 175 Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu Arg Phe Lys180 185 190 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala TyrVal 195 200 205 Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr PheCys Thr 210 215 220 Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Thr SerVal Thr Val 225 230 235 240 Ser

What is claimed is:
 1. A single-chain antigen-bindingpolypeptide-polyalkylene oxide conjugate, comprising a single-chainantigen-binding polypeptide comprising: (a) a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) a peptide linker linking the first and secondpolypeptides (a) and (b) into a single chain polypeptide having anantigen binding site, wherein said single-chain antigen-bindingpolypeptide is conjugated to polyalkylene oxide and wherein saidsingle-chain antigen-binding polypeptide-polyalkylene oxide conjugatehas an antigen binding affinity within a range of about one-fold toabout ten-fold of the antigen binding affinity of said single-chainantigen-binding polypeptide in its unconjugated form.
 2. A single-chainantigen-binding polypeptide-polyalkylene oxide conjugate, comprising asingle-chain antigen-binding polypeptide comprising: (a) a firstpolypeptide comprising the antigen binding portion of the variableregion of an antibody heavy or light chain; (b) a second polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; and (c) a peptide linker linking thefirst and second polypeptides (a) and (b) into a single chainpolypeptide having an antigen binding site, wherein said single-chainantigen-binding polypeptide is conjugated to polyalkylene oxide andwherein said single-chain antigen-binding polypeptide-polyalkylene oxideconjugate has an antigen binding affinity within about ten-fold of theantigen binding affinity of said single-chain antigen-bindingpolypeptide in its unconjugated form.
 3. A single-chain antigen-bindingpolypeptide-polyalkylene oxide conjugate, comprising a single-chainantigen-binding polypeptide comprising: (a) a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) a peptide linker linking the first and secondpolypeptides (a) and (b) into a single chain polypeptide having anantigen binding site, wherein said single-chain antigen-bindingpolypeptide is conjugated to polyalkylene oxide and wherein saidsingle-chain antigen-binding polypeptide-polyalkylene oxide conjugatehas an antigen binding affinity within about five-fold of the antigenbinding affinity of said single-chain antigen-binding polypeptide in itsunconjugated form.
 4. A single-chain antigen-bindingpolypeptide-polyalkylene oxide conjugate, comprising a single-chainantigen-binding polypeptide comprising: (a) a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) a peptide linker linking the first and secondpolypeptides (a) and (b) into a single chain polypeptide having anantigen binding site, wherein said single-chain antigen-bindingpolypeptide is conjugated to polyalkylene oxide and wherein saidsingle-chain antigen-binding polypeptide-polyalkylene oxide conjugatehas an antigen binding affinity within about two-fold of the antigenbinding affinity of said single-chain antigen-binding polypeptide in itsunconjugated form.
 5. A single-chain antigen-binding polypeptide capableof polyalkylene oxide conjugation, comprising: (a) a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) a peptide linker linking said first and secondpolypeptides (a) and (b) into a single chain polypeptide having anantigen binding site, wherein the single-chain antigen-bindingpolypeptide has at least one Cys residue which is capable ofpolyalkylene oxide conjugation, wherein said Cys residue is located at aposition selected from the group consisting of: (i) the amino acidposition 11, 12, 13, 14 or 15 of the light chain variable region; (ii)the amino acid position 77, 78 or 79 of the light chain variable region;(iii) the amino acid position 11, 12, 13, 14 or 15 of the heavy chainvariable region; (iv) the amino acid position 82B, 82C or 83 of theheavy chain variable region; (v) any amino acid position of the peptidelinker; (vi) adjacent to the C-terminus of polypeptide (a) or (b); and(vii) combinations thereof, wherein the polyalkylene oxide conjugatedsingle-chain antigen-binding polypeptide is capable of binding anantigen.
 6. The single-chain antigen-binding polypeptide of claim 5,wherein said Cys residue capable of polyalkylene oxide conjugation islocated at a position selected from the group consisting of: (i′) theamino acid position 77 of the light chain variable region; (ii′) theamino acid position 82B of the heavy chain variable region; (iii′) theamino acid position 3 of the peptide linker; (iv′) adjacent to theC-terminus of said polypeptide (a) or (b); and (v′) combinationsthereof.
 7. The single-chain antigen-binding polypeptide of claim 5,wherein said first polypeptide (a) comprises the antigen binding portionof the variable region of an antibody light chain and said secondpolypeptide (b) comprises the antigen binding portion of the variableregion of an antibody heavy chain.
 8. The single-chain antigen-bindingpolypeptide of claim 5, wherein the C-terminus of said secondpolypeptide (b) is the native C-terminus.
 9. The single-chainantigen-binding polypeptide of claim 5, wherein the C-terminus of saidsecond polypeptide (b) comprises a deletion of one or plurality of aminoacid residue(s), such that the remaining N-terminus amino acid residuesof the second polypeptide are sufficient for the polyalkylene oxideconjugated single-chain antigen-binding polypeptide to be capable ofbinding an antigen.
 10. The single-chain antigen-binding polypeptide ofclaim 5, wherein the C-terminus of said second polypeptide (b) comprisesan addition of one or plurality of amino acid residue(s), such that thepolyalkylene oxide conjugated single-chain antigen-binding polypeptideis capable of binding an antigen.
 11. The single-chain antigen-bindingpolypeptide of claim 5, wherein said wherein said Cys residue capable ofpolyalkylene oxide conjugation is attached to a polyalkylene oxidemoiety.
 12. The polyalkylene oxide conjugated single-chainantigen-binding polypeptide of claim 11, wherein said polyalkylene oxideconjugated single-chain antigen-binding polypeptide is conjugated to oneor plurality of peptide, lipid, nucleic acid, drug, toxin, chelator,boron addend or detectable label molecules.
 13. The polyalkylene oxideconjugated single-chain antigen-binding polypeptide of claim 11, whereinsaid polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is conjugated to a carrier having one or plurality ofpeptide, lipid, nucleic acid, drug, toxin, chelator, boron addend ordetectable label molecules bound to said carrier.
 14. A polynucleotidesequence encoding a single-chain antigen-binding polypeptide capable ofpolyalkylene oxide conjugation, comprising: (a) a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) a peptide linker linking said first and secondpolypeptides (a) and (b) into a single chain polypeptide having anantigen binding site, wherein the single-chain antigen-bindingpolypeptide has at least one Cys residue which is capable ofpolyalkylene oxide conjugation, wherein said Cys residue is located at aposition selected from the group consisting of: (i) the amino acidposition 11, 12, 13, 14 or 15 of said light chain variable region; (ii)the amino acid position 77, 78 or 79 of said light chain variableregion; (iii) the amino acid position 11, 12, 13, 14 or 15 of said heavychain variable region; (iv) the amino acid position 82B, 82C or 83 ofsaid heavy chain variable region; (v) any amino acid position of saidpeptide linker; (vi) adjacent to the C-terminus of said polypeptide (a)or (b); and (vii) combinations thereof, and wherein the polyalkyleneoxide conjugated single-chain antigen-binding polypeptide is capable ofbinding an antigen.
 15. The polynucleotide sequence of claim 14, whereinsaid Cys residue capable of polyalkylene oxide conjugation is located ata position selected from the group consisting of: (i′) the amino acidposition 77 of the light chain variable region; (ii′) the amino acidposition 82B of the heavy chain variable region; (iii′) the amino acidposition 3 of the peptide linker; (iv′) adjacent to the C-terminus ofsaid polypeptide (a) or (b); and (v′) combinations thereof.
 16. Areplicable cloning or expression vehicle comprising the polynucleotidesequence of claim
 14. 17. The vehicle of claim 16 which is a plasmid.18. A host cell transformed with the polynucleotide of claim
 17. 19. Thehost cell of claim 18 which is a bacterial cell, a yeast cell or otherfungal cell, an insect cell or a mammalian cell line.
 20. The host cellof claim 19 which is Pichia pastoris.
 21. A method of a producingsingle-chain antigen-binding polypeptide-polyalkylene oxide conjugate,comprising a single-chain antigen-binding polypeptide comprising: (a)providing a first genetic sequence encoding a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) providing a second genetic sequenceencoding a second polypeptide comprising the antigen binding portion ofthe variable region of an antibody heavy or light chain; and (c) linkingthe first and second genetic sequences (a) and (b) with a third geneticsequence encoding a linker into a fourth genetic sequence encoding asingle chain polypeptide having an antigen binding site, wherein saidsingle-chain antigen-binding polypeptide is capable of polyalkyleneoxide conjugation and wherein said single-chain antigen-bindingpolypeptide-polyalkylene oxide conjugate retains antigen bindingaffinity within a range ofabout one-fold to about ten-fold of theantigen binding affinity of said single-chain antigen-bindingpolypeptide in its unconjugated form.
 22. A method of producing asingle-chain antigen-binding polypeptide capable of polyalkylene oxideconjugation, comprising: (a) providing a first genetic sequence encodinga first polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; (b) providing asecond genetic sequence encoding a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) linking said first and second genetic sequences (a)and (b) with a third genetic sequence encoding a peptide linker into afourth genetic sequence encoding a single chain polypeptide having anantigen binding site, wherein the single-chain antigen-bindingpolypeptide has at least one Cys residue which is capable ofpolyalkylene oxide conjugation, wherein said Cys residue is located at aposition selected from the group consisting of: (i) the amino acidposition 11, 12, 13, 14 or 15 of said light chain variable region; (ii)the amino acid position 77, 78 or 79 of said light chain variableregion; (iii) the amino acid position 11, 12, 13, 14 or 15 of said heavychain variable region; (iv) the amino acid position 82B, 82C or 83 ofsaid heavy chain variable region; (v) any amino acid position of saidpeptide linker; (vi) adjacent to the C-terminus of said polypeptide (a)or (b); and (vii) combinations thereof, and wherein the polyalkyleneoxide conjugated single-chain antigen-binding polypeptide is capable ofbinding an antigen; (d) transforming a host cell with said fourthgenetic sequence encoding said single-chain antigen-binding polypeptideof step (c); and (e) expressing said single-chain antigen-bindingpolypeptide of step (c) in said host, thereby producing a single-chainantigen-binding polypeptide capable of polyalkylene oxide conjugation.23. The method of claim 22, wherein said Cys residue capable ofpolyalkylene oxide conjugation is located at a position selected fromthe group consisting of: (i′) the amino acid position 77 of the lightchain variable region; (ii′) the amino acid position 82B of the heavychain variable region; (iii′) the amino acid position 3 of the peptidelinker; (iv′) adjacent to the C-terminus of said polypeptide (a) or (b);and (v′) combinations thereof.
 24. The method of claim 22, said firstgenetic sequence encoding a first polypeptide (a) comprises the antigenbinding portion of the variable region of an antibody light chain andsaid second genetic sequence encoding a second polypeptide (b) comprisesthe antigen binding portion of the variable region of an antibody heavychain.
 25. The method of claim 22, wherein the C-terminus of said secondpolypeptide (b) is the native C-terminus.
 26. The method of claim 22,wherein the C-terminus of said second polypeptide (b) comprises adeletion of one or plurality of amino acid residue(s), such that theremaining N-terminus amino acid residues of the second polypeptide aresufficient for the polyalkylene oxide conjugated single-chainantigen-binding polypeptide to be capable of binding an antigen.
 27. Themethod of claim 22, wherein the C-terminus of said second polypeptide(b) comprises an addition of one or plurality of amino acid residue(s),such that the polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is capable of binding an antigen.
 28. A multivalentsingle-chain antigen-binding polypeptide-polyalkylene oxide conjugate,comprising two or more single-chain antigen binding polypeptides, eachsingle-chain antigen-binding polypeptide comprising: (a) a firstpolypeptide comprising the antigen binding portion of the variableregion of an antibody heavy or light chain; (b) a second polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; and (c) a peptide linker linking thefirst and second polypeptides (a) and (b) into a single chainpolypeptide having an antigen binding site, wherein said single-chainantigen-binding polypeptide is conjugated to polyalkylene oxide andwherein said single-chain antigen-binding polypeptide-polyalkylene oxideconjugate retains antigen binding affinity within a range ofaboutone-fold to about ten-fold of the antigen binding affinity of saidsingle-chain antigen-binding polypeptide in its unconjugated form.
 29. Amultivalent single-chain antigen-binding protein, comprising two or moresingle-chain antigen-binding polypeptides, each single-chainantigen-binding polypeptide comprising: (a) a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) a peptide linker linking said first and secondpolypeptides wherein one of the two single-chain antigen-bindingpolypeptide has at least one Cys residue which is capable ofpolyalkylene oxide conjugation, wherein said Cys residue is located at aposition selected from the group consisting of: (i) the amino acidposition 11, 12, 13, 14 or 15 of said light chain variable region; (ii)the amino acid position 77, 78 or 79 of said light chain variableregion; (iii) the amino acid position 11, 12, 13, 14 or 15 of said heavychain variable region; (iv) the amino acid position 82B, 82C or 83 ofsaid heavy chain variable region; (v) any amino acid position of saidpeptide linker; (vi) adjacent to the C-terminus of said polypeptide (a)or (b); and (vii) combinations thereof, and wherein the polyalkyleneoxide conjugated single-chain antigen-binding polypeptide is capable ofbinding an antigen.
 30. The multivalent protein of claim 29, whereinsaid Cys residue capable of polyalkylene oxide conjugation is located ata position selected from the group consisting of: (i′) the amino acidposition 77 of the light chain variable region; (ii′) the amino acidposition 82B of the heavy chain variable region; (iii′) the amino acidposition 3 of the peptide linker; (iv′) adjacent to the C-terminus ofsaid polypeptide (a) or (b); and (v′) combinations thereof.
 31. Themultivalent protein of claim 29, wherein said first polypeptide (a)comprises the antigen binding portion of the variable region of anantibody light chain and said second polypeptide (b) comprises theantigen binding portion of the variable region of an antibody heavychain.
 32. The multivalent protein of claim 29, wherein the C-terminusof said second polypeptide (b) is the native C-terminus.
 33. Themultivalent protein of claim 29, wherein the C-terminus of said secondpolypeptide (b) comprises a deletion of one or plurality of amino acidresidue(s), such that the remaining N-terminus amino acid residues ofthe second polypeptide are sufficient for the polyalkylene oxideconjugated single-chain antigen-binding polypeptide to be capable ofbinding an antigen.
 34. The multivalent protein of claim 29, wherein theC-terminus of said second polypeptide comprises an addition of one orplurality of amino acid residue(s), such that the polyalkylene oxideconjugated single-chain antigen-binding polypeptide is capable ofbinding an antigen.
 35. The multivalent protein of claim 29, whereinsaid wherein said Cys residue capable of polyalkylene oxide conjugationis attached to a polyalkylene oxide moiety.
 36. The polyalkylene oxideconjugated multivalent protein of claim 35, wherein said polyalkyleneoxide conjugated multivalent protein is conjugated to one or pluralityof peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend ordetectable label molecule(s).
 37. A method of detecting an antigensuspected of being in a sample, comprising: (a) contacting said samplewith the polyalkylene oxide conjugated polypeptide or protein of claim 1or 11, wherein said polyalkylene oxide conjugated polypeptide or proteinis conjugated to one or plurality of detectable label molecule(s), orconjugated to a carrier having one or plurality of detectable labelmolecule(s) bound to said carrier; and (b) detecting whether saidpolyalkylene oxide conjugated single-chain antigen-binding polypeptideor protein has bound to said antigen.
 38. A method of imaging theinternal structure of an animal, comprising administering to said animalan effective amount of the polyalkylene oxide conjugated polypeptide orprotein of claim 1 or 11, wherein said polyalkylene oxide conjugatedpolypeptide or protein is conjugated to one or plurality of detectablelabel or chelator molecule(s), or conjugated to a carrier having one orplurality of detectable label or chelator molecule(s) bound to saidcarrier, and measuring detectable radiation associated with said animal.39. The method of claim 38, wherein said animal includes a human.
 40. Amethod for treating a targeted disease, comprising administering aneffective amount of a composition comprising the polyalkylene oxideconjugated polypeptide or protein of claim 1 or 11 and apharmaceutically acceptable carrier vehicle, wherein said polyalkyleneoxide conjugated polypeptide or protein is conjugated to one orplurality of peptide, lipid, nucleic acid, drug, toxin, boron addend orradioisotope molecule(s), or conjugated to a carrier having one orplurality of peptide, lipid, nucleic acid, drug, toxin, boron addend orradioisotope molecule(s) bound to said carrier.
 41. A single-chainantigen-binding polypeptide capable of polyalkylene oxide conjugation,comprising: (a) a first polypeptide comprising the antigen bindingportion of the variable region of an antibody heavy or light chain; (b)a second polypeptide comprising the antigen binding portion of thevariable region of an antibody heavy or light chain; and (c) a peptidelinker linking the first and second polypeptides (a) and (b) into asingle chain polypeptide having an antigen binding site, wherein thesingle-chain antigen-binding polypeptide has at least three consecutiveLys residues which are capable of polyalkylene oxide conjugation andwherein any one of said consecutive Lys residues is located at aposition selected from the group consisting of (i) any amino acidposition of the peptide linker; (ii) adjacent to the C-terminus of thesecond polypeptide (b); and (iii) combinations thereof, wherein thepolyalkylene oxide conjugated single-chain antigen-binding polypeptideis capable of binding an antigen.
 42. A polynucleotide sequence encodinga single-chain antigen-binding polypeptide capable of polyalkylene oxideconjugation, comprising: (a) a first polypeptide comprising the antigenbinding portion of the variable region of an antibody heavy or lightchain; (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and (c) apeptide linker linking the first and second polypeptides (a) and (b)into a single chain polypeptide having an antigen binding site, whereinthe single-chain antigen-binding polypeptide has at least threeconsecutive Lys residues which are capable of polyalkylene oxideconjugation and wherein any one of said consecutive Lys residues islocated at a position selected from the group consisting of (i) anyamino acid position of the peptide linker; (ii) adjacent to theC-terminus of the second polypeptide (b); and (iii) combinationsthereof, wherein the polyalkylene oxide conjugated single-chainantigen-binding polypeptide is capable of binding an antigen.
 43. Areplicable cloning or expression vehicle comprising the polynucleotidesequence of claim
 42. 44. The vehicle of claim 43 which is a plasmid.45. A host cell transformed with the polynucleotide of claim
 42. 46. Thehost cell of claim 39 which is a bacterial cell, a yeast cell or otherfungal cell, an insect cell or a mammalian cell line.
 47. The host cellof claim 46 which is Pichia pastoris.
 48. A method of producing asingle-chain antigen-binding polypeptide capable of polyalkylene oxideconjugation, comprising: (a) a first polypeptide comprising the antigenbinding portion of the variable region of an antibody heavy or lightchain; (b) a second polypeptide comprising the antigen binding portionof the variable region of an antibody heavy or light chain; and (c) apeptide linker linking the first and second polypeptides (a) and (b)into a single chain polypeptide having an antigen binding site, whereinthe single-chain antigen-binding polypeptide has at least threeconsecutive Lys residues which are capable of polyalkylene oxideconjugation and wherein any one of said consecutive Lys residues islocated at a position selected from the group consisting of (i) anyamino acid position of the peptide linker; (ii) adjacent to theC-terminus of the second polypeptide (b); and (iii) combinationsthereof, wherein the polyalkylene oxide conjugated single-chainantigen-binding polypeptide is capable of binding an antigen.
 49. Thesingle-chain antigen-binding polypeptide of claim 41, wherein saidwherein said wherein any one of said consecutive Lys residues capable ofpolyalkylene oxide conjugation is attached to a polyalkylene oxidemoiety.
 50. The polyalkylene oxide conjugated single-chainantigen-binding polypeptide of claim 49, wherein said polyalkylene oxideconjugated single-chain antigen-binding polypeptide is conjugated to oneor plurality of peptide, lipid, nucleic acid, drug, toxin, chelator,boron addend or detectable label molecules.
 51. The polyalkylene oxideconjugated single-chain antigen-binding polypeptide of claim 49, whereinsaid polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is conjugated to a carrier having one or plurality ofpeptide, lipid, nucleic acid, drug, toxin, chelator, boron addend ordetectable label molecules bound to said carrier.
 52. A method ofproducing a single-chain antigen-binding polypeptide capable ofpolyalkylene oxide conjugation, comprising: (a) providing a firstgenetic sequence encoding a first polypeptide comprising the antigenbinding portion of the variable region of an antibody heavy or lightchain; (b) providing a second genetic sequence encoding a secondpolypeptide comprising the antigen binding portion of the variableregion of an antibody heavy or light chain; and (c) linking said firstand second genetic sequences (a) and (b) with a third genetic sequenceencoding a peptide linker into a fourth genetic sequence encoding asingle chain polypeptide having an antigen binding site, wherein thesingle-chain antigen-binding polypeptide has at least three consecutiveLys residues which are capable of polyalkylene oxide conjugation andwherein any one of said consecutive Lys residues is located at aposition selected from the group consisting of (i) any amino acidposition of the peptide linker; (ii) adjacent to the C-terminus of thesecond polypeptide (b); and (iii) combinations thereof, wherein thepolyalkylene oxide conjugated single-chain antigen-binding polypeptideis capable of binding an antigen; (d) transforming a host cell with saidfourth genetic sequence encoding said single-chain antigen-bindingpolypeptide of step (c); and (e) expressing said single-chainantigen-binding polypeptide of step (c) in said host, thereby producinga single-chain antigen-binding polypeptide capable of polyalkylene oxideconjugation.
 53. The method of claim 52, said first genetic sequenceencoding a first polypeptide (a) comprises the antigen binding portionof the variable region of an antibody light chain and said secondgenetic sequence encoding a second polypeptide (b) comprises the antigenbinding portion of the variable region of an antibody heavy chain. 54.The method of claim 52, wherein the C-terminus of said secondpolypeptide (b) is the native C-terminus.
 55. The method of claim 52,wherein the C-terminus of said second polypeptide (b) comprises adeletion of one or plurality of amino acid residue(s), such that theremaining N-terminus amino acid residues of the second polypeptide aresufficient for the polyalkylene oxide conjugated single-chainantigen-binding polypeptide to be capable of binding an antigen.
 56. Themethod of claim 52, wherein the C-terminus of said second polypeptide(b) comprises an addition of one or plurality of amino acid residue(s),such that the polyalkylene oxide conjugated single-chain antigen-bindingpolypeptide is capable of binding an antigen.
 57. A multivalentsingle-chain antigen-binding protein, comprising two or moresingle-chain antigen-binding polypeptides, each single-chainantigen-binding polypeptide comprising: (a) a first polypeptidecomprising the antigen binding portion of the variable region of anantibody heavy or light chain; (b) a second polypeptide comprising theantigen binding portion of the variable region of an antibody heavy orlight chain; and (c) a peptide linker linking said first and secondpolypeptides wherein one of the two single-chain antigen-bindingpolypeptides has at least three consecutive Lys residues which arecapable of polyalkylene oxide conjugation and wherein any one of saidconsecutive Lys residues is located at a position selected from thegroup consisting of (i) any amino acid position of the peptide linker;(ii) adjacent to the C-terminus of the second polypeptide (b); and (iii)combinations thereof, wherein the polyalkylene oxide conjugatedsingle-chain antigen-binding polypeptide is capable of binding anantigen.
 58. The multivalent protein of claim 57, wherein said firstpolypeptide (a) comprises the antigen binding portion of the variableregion of an antibody light chain and said second polypeptide (b)comprises the antigen binding portion of the variable region of anantibody heavy chain.
 59. The multivalent protein of claim 57, whereinthe C-terminus of said second polypeptide (b) is the native C-terminus.60. The multivalent protein of claim 57, wherein the C-terminus of saidsecond polypeptide (b) comprises a deletion of one or plurality of aminoacid residue(s), such that the remaining N-terminus amino acid residuesof the second polypeptide are sufficient for the polyalkylene oxideconjugated single-chain antigen-binding polypeptide to be capable ofbinding an antigen.
 61. The multivalent protein of claim 57, wherein theC-terminus of said second polypeptide comprises an addition of one orplurality of amino acid residue(s), such that the polyalkylene oxideconjugated single-chain antigen-binding polypeptide is capable ofbinding an antigen.
 62. The multivalent protein of claim 57, wherein anyone of said consecutive Lys residues capable of polyalkylene oxideconjugation is attached to a polyalkylene oxide moiety.
 63. Thepolyalkylene oxide conjugation multivalent protein of claim 62, whereinsaid polyalkylene oxide conjugated multivalent protein is conjugated toone or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator,boron addend or detectable label molecule(s).